Methods for generating stably linked complexes composed of homodimers, homotetramers or dimers of dimers and uses

ABSTRACT

The present invention concerns methods and compositions for stably tethered structures of defined compositions, which may have multiple functionalities and/or binding specificities. Particular embodiments concern homodimers comprising monomers that contain a dimerization and docking domain attached to a precursor. The precursors may be virtually any molecule or structure, such as antibodies, antibody fragments, antibody analogs or mimetics, aptamers, binding peptides, fragments of binding proteins, known ligands for proteins or other molecules, enzymes, detectable labels or tags, therapeutic agents, toxins, pharmaceuticals, cytokines, interleukins, interferons, radioisotopes, proteins, peptides, peptide mimetics, polynucleotides, RNAi, oligosaccharides, natural or synthetic polymeric substances, nanoparticles, quantum dots, organic or inorganic compounds, etc. Other embodiments concern tetramers comprising a first and second homodimer, which may be identical or different. The disclosed methods and compositions provide a facile and general way to obtain homodimers, homotetramers and heterotetramers of virtually any functionality and/or binding specificity.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/389,358, filed Mar. 24, 2006, which claimed the benefit under 35U.S.C. §119(e) of provisional U.S. patent application Ser. No.60/668,603, filed Apr. 6, 2005; 60/728,292, filed Oct. 20, 2005;60/751,196, filed Dec. 16, 2005; and 60/782,332, filed Mar. 14, 2006.The text of each of the priority applications is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Man-made agents that incorporate multiple copies of both targeting andeffector moieties are highly desirable, as they should provide more avidbinding and confer enhanced potency. Although recombinant technologiesare commonly applied for making fusion proteins with both targeting andeffector domains, multimeric structures that comprise the same ordifferent monomeric components to acquire multivalency ormultifunctionality may be obtained only with judicious applications ofconjugation chemistries.

For agents generated by recombinant engineering, problems may includehigh manufacturing cost, low expression yields, instability in serum,instability in solution resulting in formation of aggregates ordissociated subunits, undefined batch composition due to the presence ofmultiple product forms, contaminating side-products, reduced functionalactivities or binding affinity/avidity attributed to steric factors oraltered conformations, etc. For agents generated by various methods ofchemical cross-linking, high manufacturing cost and heterogeneity of thepurified product are two major limitations.

Thus, there remains a need in the art for a method of making multivalentstructures of multiple specificities or functionalities in general,which are of defined composition, homogeneous purity, and unalteredaffinity, and can be produced in high yields without the requirement ofextensive purification steps. Furthermore, such structures must also besufficiently stable in serum to allow in vivo applications. A needexists for stable, multivalent structures of multiple specificities orfunctionalities that are easy to construct and/or obtain in relativelypurified form.

SUMMARY OF THE INVENTION

The present invention discloses a platform technology for generatingstably tethered structures that may have multiple functions or bindingspecificities or both, and are suitable for in vitro as well as in vivoapplications. In one embodiment, the stably tethered structures areproduced as a homodimer of any organic substance, which can be proteinsor non-proteins. The homodimer, referred to as a₂ hereafter, is composedof two identical subunits linked to each other via a distinct peptidesequence, termed the dimerization and docking domain (DDD), which iscontained in each subunit. The subunit is constructed by linking a DDDsequence to a precursor of interest by recombinant engineering orchemical conjugation via a spacer group, resulting in a structure thatis capable of self-association to form a dimer. Representative a₂constructs made with the DDD sequence referred to as DDD1 (FIG. 1 a, SEQID NO:1) are described in Examples 2 and 3.

In another embodiment, the stably tethered structures are producedpredominantly as a homotetramer of any organic substance, which can beproteins or non-proteins. The homotetramer, referred to as a₄ hereafter,is composed of two identical a₂ constructs made with the DDD sequencereferred to as DDD2 (FIG. 1 b, SEQ ID NO:2), which is contained in eachof the four subunits. Five such a₄ constructs are described in Examples4 and 5.

In yet another embodiment, the stably tethered structures are producedas a hybrid tetramer from any two distinct a₄ constructs. The hybridtetramer, referred to as a₂a′₂ hereafter, is composed of two differenta₂ constructs derived from respective a₄ constructs. Three such a₂a′₂constructs are described in Example 6. In other embodiments, fusionproteins that are single-chain polypeptides comprising multiple domains,such as avimers (Silverman et al., Nat. Biotechnol. (2005), 23:1556-1561) for example, may serve as precursors of interest to increasethe valency, functionality, and specificity of the resulting a₂, a₄ anda₂a′₂ constructs, which may be further conjugated with effectors andcarriers to acquire additional functions enabled by such modifications.

Numerous a₂, a₄ and a₂a′₂ constructs can be designed and produced withthe disclosed methods and compositions. For example, at least 7 types ofprotein- or peptide-based constructs as listed below are envisioned:

-   Type 1: A bivalent a₂ construct composed of two Fab or scFv    fragments derived from the same mAb. See Table 1 for selected    examples.-   Type 2: A bivalent a₂ construct composed of two identical    non-immunoglobulin proteins. See Table 2 for selected examples.-   Type 3: A tetravalent a₄ construct composed of four Fab or scFv    fragments derived from the same mAb. See Table 3 for selected    examples.-   Type 4: A tetravalent a₄ constructs composed of four identical    non-immunoglobulin proteins. See Table 4 for selected examples.-   Type 5: A bispecific tetravalent a₂a′₂ construct composed of two Fab    or scFv fragments derived from the same mAb and two Fab or scFv    fragment derived from a different mAb. See Table 5 for selected    examples.-   Type 6: A multifunctional a₂a′₂ constructs composed of two Fab or    scFv fragments derived from the same mAb and two identical    non-immunoglobulin proteins. See Table 6 for selected examples.-   Type 7: A multifunctional a₂a′₂ constructs composed of two pairs of    different non-immunoglobulin proteins. See Table 7 for selected    examples.

In general, the products in the type 1 category are useful in variousapplications where a bivalent binding protein composed of two stablytethered Fab (or scFv) fragments derived from the same monoclonalantibody is more desirable than the corresponding bivalent F(ab′)₂,which is known to dissociate into monovalent Fab′ in vivo. For example,the efficacy of an a₂ product composed of two stably tethered Fabfragments of 7E3 should be improved over that of ReoPro™ (Centocor,Inc.), which uses the Fab fragment of 7E3 to prevent plateletaggregation.

In general, the products in the type 2 category are useful in variousapplications where a bivalent agent may be more desirable than amonovalent agent either for improved efficacy or pharmacokinetics orboth. For example, an a₂ product composed of two copies oferythropoietin may be preferred to Epogen® (Amgen), which contains onlyone erythropoietin. Another example is an a₂ product composed of twocopies of Aβ12-28P fused to the CH2 and CH3 domains of human IgG1.Aβ12-28P is the peptide containing the N-terminal 12 to 28 residues ofβ-amyloid (Aβ) with valine at position 18 replaced by proline. Aβ12-28Pis non-fibrillogenic and nontoxic and can block the binding ofapolipoprotein E (apoE) to Aβ with reduction of Aβ plaques in atransgenic mouse model (Sadowski et al., Am J Pathol. (2004), 165:937-948). The fusion of CH2 and CH3 to Aβ12-28P would serve twopurposes: (1) to facilitate the resulting complex to cross the bloodbrain barrier through the FcRn; (2) for effective reduction of Aβ plaqueby microglia cells following binding of Aβ to the anti-Aβ arms andbinding of the CH2-CH3 domain to the Fc receptors on microglia (Hartmanet al., J. Neurosci. (2005), 25: 6213-6220).

In general, the products in the type 3 category are useful in variousapplications where a tetravalent binding protein composed of four stablytethered Fab (or scFv) fragments derived from the same monoclonalantibody is more desirable than a trivalent, bivalent or monovalentbinding protein based on the same monoclonal antibody. For example, theefficacy of an a₄ product composed of four stably tethered Fab fragmentsof an anti-TNF-α antibody such as adalimumab may be more efficacious intreating arthritis than HUMIRA™ (Abbott Laboratories).

In general, the products in the type 4 category are useful in variousapplications where a tetravalent agent may be more desirable than atrivalent, bivalent or monovalent agent due to the enhanced avidity ofbinding to the target. For example, an a₄ product composed of fourcopies of factor IX may be preferred as a therapeutic agent for treatinghemophilia to Benefix™ (Wyeth), which contains only one factor IX.

In general, the products in the type 5 category are useful in variousapplications where a bispecific tetravalent binding protein composed oftwo different a₂ subunits is desired. For example, an a₂a′₂ productcomposed of two Fab fragments of trastuzumab and two Fab fragments ofpertuzumab may be more efficacious than either Herceptin® (Genentech) orOmnitarg™ (Genentech) for treating cancers that overexpress the HER2receptor.

In general, the products in the type 6 category are useful in variousapplications where target-specific delivery or binding of anon-immunoglobulin protein is desired. For example, an a₂a′₂ productcomposed of two Fab fragments of an internalizing antibody against atumor associated antigen (such as CD74) and two copies of a toxin (suchas deglycosylated ricin A chain or ranpirnase) would be valuable forselective delivery of the toxin to destroy the target tumor cell.Another example is an a₂a′₂ product composed of two Fab fragments of anantibody against Aβ and two copies of transferin (Tf), which is expectedto cross the blood brain barrier and neutralize Aβ for effective therapyof Alzheimer's disease.

In general, the products in the type 7 category are useful in variousapplications where the combination of two different non-immunoglobulinproteins are more desirable than each respective non-immunoglobulinprotein alone. For example, an a₂a′₂ product composed of two copies of asoluble component of the receptor for IL-4R (sIL-4R) and two copies of asoluble component of the receptor for IL-13 (sIL-13R) would be apotential therapeutic agent for treating asthma or allergy. Anotherexample is an a₂a′₂ product composed of two copies of Aβ12-28P and twocopies of Tf. The addition of Tf to Aβ12-28P is expected to enable theresulting complex to cross the blood brain barrier for effectivetreatment of Alzheimer's disease.

The stably tethered structures of the present invention, including theirconjugates, are suitable for use in a wide variety of therapeutic anddiagnostic applications. For example, the a₂, a₄, or a₂a′₂ constructsbased on the antibody binding domains can be used for therapy where sucha construct is not conjugated to an additional functional agent, in thesame manner as therapy using a naked antibody. Alternatively, thesestably tethered structures can be derivatized with one or morefunctional agents to enable diagnostic or therapeutic applications. Theadditional agent may be covalently linked to the stably tetheredstructures using conventional conjugation chemistries.

Methods of use of stably tethered structures may include detection,diagnosis and/or treatment of a disease or other medical condition. Suchconditions may include, but are not limited to, cancer, hyperplasia,diabetic retinopathy, macular degeneration, inflammatory bowel disease,Crohn's disease, ulcerative colitis, rheumatoid arthritis, sarcoidosis,asthma, edema, pulmonary hypertension, psoriasis, corneal graftrejection, neovascular glaucoma, Osler-Webber Syndrome, myocardialangiogenesis, plaque neovascularization, restenosis, neointima formationafter vascular trauma, telangiectasia, hemophiliac joints, angiofibroma,fibrosis associated with chronic inflammation, lung fibrosis, deepvenous thrombosis or wound granulation.

In particular embodiments, the disclosed methods and compositions may beof use to treat autoimmune disease, such as acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, juvenile diabetes mellitus,Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosum,Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiplesclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgAnephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture'ssyndrome, thromboangitisubiterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In certain embodiments, the stably tethered structures may be of use fortherapeutic treatment of cancer. It is anticipated that any type oftumor and any type of tumor antigen may be targeted. Exemplary types oftumors that may be targeted include acute lymphoblastic leukemia, acutemyelogenous leukemia, biliary cancer, breast cancer, cervical cancer,chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectalcancer, endometrial cancer, esophageal, gastric, head and neck cancer,Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin'slymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreaticcancer, glioma, melanoma, liver cancer, prostate cancer, and urinarybladder cancer.

Tumor-associated antigens that may be targeted include, but are notlimited to, carbonic anhydrase IX, A3, antigen specific for A33antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA 95,NCA90, HCG and its subunits, CEA (CEACAM-5), CEACAM-6, CSAp, EGFR,EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia inducible factor (HIF),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibition factor(MIF), MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4-antigen, PSA, PSMA, RS5,S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growth factor-1(IGF-1), Tn antigen, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGF, placenta growth factor (P1GF), 17-1A-antigen, anangiogenesis marker (e.g., ED-B fibronectin), an oncogene marker, anoncogene product, and other tumor-associated antigens. Recent reports ontumor associated antigens include Mizukami et al., (2005, Nature Med.11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets 5:229-48);Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Ren et al.(2005, Ann. Surg. 242:55-63), each incorporated herein by reference.

In other embodiments, the stably tethered structures may be of use totreat infection with pathogenic organisms, such as bacteria, viruses orfungi. Exemplary fungi that may be treated include Microsporum,Trichophyton, Epidermophyton, Sporothrix schenckii, Cryptococcusneoformans, Coccidioides immitis, Histoplasma capsulatum, Blastomycesdermatitidis or Candida albican. Exemplary viruses include humanimmunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabiesvirus, influenza virus, human papilloma virus, hepatitis B virus,hepatitis C virus, Sendai virus, feline leukemia virus, Reo virus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus or blue tongue virus. Exemplary bacteria includeBacillus anthraces, Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus spp., Hemophilis influenzae B,Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis or aMycoplasma.

Although not limiting, in various embodiments, the precursorsincorporated into the monomers, dimers and/or tetramers may comprise oneor more proteins, such as a bacterial toxin, a plant toxin, ricin,abrin, a ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonasexotoxin, Pseudomonas endotoxin, Ranpirnase (Rap), Rap (N69Q), PE38,dgA, DT390, PLC, tPA, a cytokine, a growth factor, a soluble receptorcomponent, surfactant protein D, IL-4, sIL-4R, sIL-13R, VEGF₁₂₁, TPO,EPO, a clot-dissolving agent, an enzyme, a fluorescent protein, sTNFα-R,an avimer, a scFv, a dsFv or a nanobody.

In other embodiments, an anti-angiogenic agent may form part or all of aprecursor or may be attached to a stably tethered structure. Exemplaryanti-angiogenic agents of use include angiostatin, baculostatin,canstatin, maspin, anti-VEGF antibodies or peptides, anti-placentalgrowth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Flt-1antibodies or peptides, laminin peptides, fibronectin peptides,plasminogen activator inhibitors, tissue metalloproteinase inhibitors,interferons, interleukin 12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2 ,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline.

In still other embodiments, one or more therapeutic agents, such asaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, CELEBREX®, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, VELCADE®, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, ONCONASE®, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, anantisense oligonucleotide, an interference RNA, or a combinationthereof, may be conjugated to or incorporated into a stably tetheredstructure.

In various embodiments, one or more effectors, such as a diagnosticagent, a therapeutic agent, a chemotherapeutic agent, a radioisotope, animaging agent, an anti-angiogenic agent, a cytokine, a chemokine, agrowth factor, a drug, a prodrug, an enzyme, a binding molecule, aligand for a cell surface receptor, a chelator, an immunomodulator, anoligonucleotide, a hormone, a photodetectable label, a dye, a peptide, atoxin, a contrast agent, a paramagnetic label, an ultrasound label, apro-apoptotic agent, a liposome, a nanoparticle or a combinationthereof, may be attached to a stably tethered structure.

Various embodiments may concern stably tethered structures and methodsof use of same that are of use to induce apoptosis of diseased cells.Further details may be found in U.S. Patent Application Publication No.20050079184, the entire text of which is incorporated herein byreference. Such structures may comprise a first and/or second precursorwith binding affinity for an antigen selected from the group consistingof CD2, CD3, CD8, CD10, CD21, CD23, CD24, CD25, CD30, CD33, CD37, CD38,CD40, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147,HLA-DR, CEA, CSAp, CA-125, TAG-72, EFGR, HER2, HER3, HER4, IGF-1R,c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, Fas (CD95),DR3, DR4, DR5, DR6, VEGF, PIGF, ED-B fibronectin, tenascin, PSMA, PSA,carbonic anhydrase IX, and IL-6. In more particular embodiments, astably tethered structure of use to induce apoptosis may comprisemonoclonal antibodies, Fab fragments, chimeric, humanized or humanantibodies or fragments. In preferred embodiments, the stably tetheredstructure may comprise combinations of anti-CD74 X anti-CD20, anti-CD74X anti-CD22, anti-CD22 X anti-CD20, anti-CD20 X anti-HLA-DR, anti-CD19 Xanti-CD20, anti-CD20 X anti-CD80, anti-CD2 X anti-CD25, anti-CD8 Xanti-CD25, and anti-CD2 X anti-CD147. In more preferred embodiments, thechimeric, humanized or human antibodies or antibody fragments may bederived from the variable domains of LL2 (anti-CD22), LL1 (anti-CD74)and A20 (anti-CD20).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two exemplary DDD sequences. The underlined sequence inDDD1 (SEQ ID NO:1) corresponds to the first 44 amino-terminal residuesfound in the RIIα of human PKA. DDD2 (SEQ ID NO:2) differs from DDD1 inthe two amino acid residues at the N-terminus.

FIG. 2 shows a schematic diagram for pdHL2-based expression vectors forIgG (upper panel) and C-DDD1-Fab (lower panel).

FIG. 3 shows a schematic diagram of C-DDD1-Fab-hMN-14 and the putativea₂ structure formed by DDD1 mediated dimerization.

FIG. 4 shows a schematic diagram of N-DDD1-Fab-hMN-14 and the putativea₂ structure formed by DDD1 mediated dimerization.

FIG. 5 shows the peptide sequence for AD1-C (SEQ ID NO:3).

FIG. 6 shows SE-HPLC analysis of affinity-purified C-DDD1-Fab-hMN-14.

FIG. 7 shows SDS-PAGE analysis of affinity-purified C-DDD1-Fab-hMN-14.

FIG. 8 shows SE-HPLC analysis of affinity-purified N-DDD1-Fab-hMN-14.

FIG. 9 shows C-DDD1-Fab-hMN-14 contains two active binding sites.

FIG. 10 shows N-DDD1-Fab-hMN-14 contains two active binding sites

FIG. 11 shows the binding affinity of C-DDD1-Fab-hMN-14 is at leastequivalent to the bivalent hMN-14 IgG or F(ab′)2 and about 5-fold higherthan the monovalent Fab.

FIG. 12 shows the binding affinity of N-DDD1-Fab-hMN-14 is equivalent tobivalent hMN-14 IgG and the binding affinity of C-DDD1-Fab-hMN-14 ishigher than hMN-14 IgG.

FIG. 13 shows C-DDD1-Fab-hMN-14 is stable in pooled human serum with noapparent change in molecular integrity over 96 h.

FIG. 14 shows C-DDD1-Fab-hMN-14 is stable in pooled human serum withunchanged immunoreactivity over 28 h.

FIG. 15 compares the tumor uptake of C-DDD1-Fab-hMN-14 with that ofhBS14-1 in mice bearing human colorectal cancer xenografts.

FIG. 16 compares the normal organ uptake of C-DDD1-Fab-hMN-14 with thatof hBS14-1 in mice bearing human colorectal cancer xenografts

FIG. 17 shows the SE-HPLC analysis of affinity-purifiedRap-hPAM4-Fab-DDD1.

FIG. 18 shows the binding affinity of Rap-hPAM4-Fab-DDD1 is equivalentto that of hPAM4 IgG.

FIG. 19 shows the predominant presence of the a₄ form inN-DDD2-Fab-hMN-14 purified with CBind L (Protein L cellulose). TheSE-HPLC trace also reveals the presence of the a₂ form, as well as freelight chains in both monomeric and dimeric forms.

FIG. 20 shows the dissociation of the a₄ form present in purifiedN-DDD2-Fab-hMN-14 to the a₂ form upon reduction with 5 mM TCEP, whichalso converts the dimeric light chain to monomeric light chain.

FIG. 21 shows a schematic representation of the conversion ofC-DDD2-Fab-hMN-14 in the a₄ form to the a₂ form upon reduction.

FIG. 22 shows the SE-HPLC analysis of the tetravalent C-DDD2-Fab-hMN-14after purification by Superdex-200 gel filtration chromatography. Twocolumns (Biosil SEC 250) were connected in tandem for increasedresolution. The tetravalent C-DDD2-Fab-hMN-14 appears as a single peak(indicated as A₄) with a retention time of 19.58 min.

FIG. 23 shows that the tetravalent C-DDD2-Fab-hMN-14 consists of fourfunctional CEA-binding Fab fragments. The conditions of SE-HPLC were thesame as described for FIG. 22. (a) When WI2 Fab′ was mixed with thetetravalent C-DDD2-Fab-hMN-14 at 1:1 molar ratio, four protein peaksrepresenting the binding of the tetravalent C-DDD2-Fab-hMN-14 to one(indicated as 1, at 18.32 min), two (indicated as 2, at 17.45 min) orthree (indicated as 3, at 16.92 min) WI2 Fab′ fragments as well as theunbound form of C-DDD2-Fab-hMN-14 were observed. (b) When WI2 Fab′ wasmixed with the tetravalent C-DDD2-Fab-hMN-14 at 5:1 molar ratio, onlythe complex consisting of the tetravalent C-DDD2-Fab-hMN-14 bound tofour WI2 Fab fragment's was observed (indicated as 4) at 16.24 min.Excess WI2 Fab′ (indicated as W) was detected at 24.17 min peak.

FIG. 24 shows SE-HPLC analysis of the tetravalent C-DDD2-Fab-hA20 afterpurification by Superdex-200 gel filtration chromatography.

FIG. 25 shows cell growth inhibition by the tetravalent C-DDD2-Fab-hA20(abbreviated as hA20A4). Daudi (1-1) cells (upper panel) or Ramos cells(lower panel) were resuspended in 48-well plates in duplicate at a finaldensity of 100,000 cells/mL in the complete medium containing 10 nM ofhA20, hA20 F(ab′)₂, or hA20A4, in the absence or presence of anti-IgM(0.1 ug/mL). The cells were incubated for 3 days and MTT assay wasperformed to determine the viable cell populations. Only hA20A4 causedsignificant growth inhibition (40 to 50%) in the absence of anti-IgM.

FIG. 26 shows the presence of the bispecific tetravalent hMN-3xhA20 byELISA.

FIG. 27 shows the presence of bispecific tetravalent hMN-3xhMN-14 byflow cytometry. BXPC3 cells, which express high levels of CEACAM6 butonly background levels of CEACAM5,were incubated for 1 h at RT with eachof the samples (10 ug/mL) in the presence of Alexa-532-WI2, a ratanti-ideotypic mAb for hMN-14 labeled with a fluorescent tag, andanalyzed by flow cytometry using Guava PCA. Only the histogram of thesample containing bispecific hMN-3xhMN-14 showed positively stainedBXPC3 cells.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety.

Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion or analog of an immunoglobulin molecule, like an antibodyfragment.

An antibody fragment is a portion of an antibody such as F(ab)₂,F(ab′)₂, Fab, Fv, sFv and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains, recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”), and minimal recognition units (CDR) consisting of theamino acid residues that mimic the hypervariable region.

An effector is an atom, molecule, or compound that brings about a chosenresult. An effector may include a therapeutic agent and/or a diagnosticagent.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, drugs, toxins, enzymes, nucleases,hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes. Other exemplary therapeutic agents and methodsof use are disclosed in U.S. Patent Publication Nos. 20050002945,20040018557, 20030148409 and 20050014207, each incorporated herein byreference.

A diagnostic agent is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An immunoconjugate is a conjugate of a binding molecule (e.g., anantibody component) with an atom, molecule, or a higher-orderedstructure (e.g., with a carrier, a therapeutic agent, or a diagnosticagent).

A naked antibody is an antibody that is not conjugated to any otheragent.

A carrier is an atom, molecule, or higher-ordered structure that iscapable of associating with a therapeutic or diagnostic agent tofacilitate delivery of such agent to a targeted cell. Carriers mayinclude lipids (e.g., amphiphilic lipids that are capable of forminghigher-ordered structures), polysaccharides (such as dextran), or otherhigher-ordered structures, such as micelles, liposomes, ornanoparticles.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent scFv or antibody fragments with the same or differentspecificities are linked. Valency of the fusion protein indicates howmany binding arms or sites the fusion protein has to a single antigen orepitope; i.e., monovalent, bivalent, trivalent or multivalent. Themultivalency of the antibody fusion protein means that it can takeadvantage of multiple interactions in binding to an antigen, thusincreasing the avidity of binding to the antigen. Specificity indicateshow many antigens or epitopes an antibody fusion protein is able tobind; i.e., monospecific, bispecific, trispecific, multispecific. Usingthese definitions, a natural antibody, e.g., an IgG, is bivalent becauseit has two binding arms but is monospecific because it binds to oneepitope. Monospecific, multivalent fusion proteins have more than onebinding site for an epitope but only binds to one such epitope, forexample a diabody with two binding site reactive with the same antigen.The fusion protein may comprise a single antibody component, amultivalent or multispecific combination of different antibodycomponents, or multiple copies of the same antibody component. Thefusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

An antibody or immunoconjugate preparation, or a composition describedherein, is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. Inparticular, an antibody preparation is physiologically significant ifits presence invokes an antitumor response or mitigates the signs andsymptoms of an autoimmune disease state. A physiologically significanteffect could also be the evocation of a humoral and/or cellular immuneresponse in the recipient mammal leading to growth inhibition or deathof target cells.

Conjugates of the Stably Tethered Structures

Additional moieties can be conjugated to the stably tethered structuresdescribed above. For example, drugs, toxins, radioactive compounds,enzymes, hormones, cytotoxic proteins, chelates, cytokines, and otherfunctional agents may be conjugated to the stably tethered structures.Conjugation can be via, for example, covalent attachments to amino acidresidues containing amine, carboxyl, thiol or hydroxyl groups in theside-chains . Various conventional linkers may be used for this purpose,for example, diisocyanates, diisothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides, maleimide-hydroxysuccinimide esters,glutaraldehyde and the like. Conjugation of agents to the stablytethered structures preferably does not significantly affect theactivity of each subunit contained in the unmodified structures.Conjugation can be carried out separately to the a₄ and a′₄ constructsand the resulting conjugates are used for preparing the a₂a′₂ constructsIn addition, cytotoxic agents may be first coupled to a polymericcarrier, which is then conjugated to a stably tethered structure. Forthis method, see Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870,1978; U.S. Pat. No. 4,699,784 and U.S. Pat. No. 4,046,722, which areincorporated herein by reference.

The conjugates described herein can be prepared by various methods knownin the art. For example, a stably tethered structure can be radiolabeledwith ¹³¹I and conjugated to a lipid, such that the resulting conjugatecan form a liposome. The liposome may incorporate one or moretherapeutic (e.g., a drug such as FUdR-dO) or diagnostic agents.Alternatively, in addition to the carrier, a stably tethered structuremay be conjugated to ¹³¹I (e.g., at a tyrosine residue) and a drug(e.g., at the epsilon amino group of a lysine residue), and the carriermay incorporate an additional therapeutic or diagnostic agent.Therapeutic and diagnostic agents may be covalently associated with oneor more than one subunit of the stably tethered structures.

The formation of liposomes and micelles is known in the art. See, e.g.,Wrobel and Collins, Biochimica et Biophysica Acta (1995), 1235: 296-304;Lundberg et al., J. Pharm. Pharmacol. (1999), 51:1099-1105; Lundberg etal., Int. J. Pharm. (2000), 205:101-108; Lundberg, J. Pharm. Sci.(1994), 83:72-75; Xu et al., Molec. Cancer Ther. (2002), 1:337-346;Torchilin et al., Proc. Nat'l. Acad. Sci., U.S.A. (2003), 100:6039-6044;U.S. Pat. No. 5,565,215; U.S. Pat. No. 6,379,698; and U.S. 2003/0082154.

Nanoparticles or nanocapsules formed from polymers, silica, or metals,which are useful for drug delivery or imaging, have been described aswell. See, e.g., West et al., Applications of Nanotechnology toBiotechnology (2000), 11:215-217; U.S. Pat. No. 5,620,708; U.S. Pat. No.5,702,727; and U.S. Pat. No. 6,530,944. The conjugation of antibodies orbinding molecules to liposomes to form a targeted carrier fortherapeutic or diagnostic agents has been described. See, e.g., Bendas,Biodrugs (2001), 15:215-224; Xu et al., Mol. Cancer Ther (2002),1:337-346; Torchilin et al., Proc. Nat'l. Acad. Sci. U.S.A (2003),100:6039-6044; Bally, et al., J. Liposome Res. (1998), 8:299-335;Lundberg, Int. J. Pharm. (1994), 109:73-81; Lundberg, J. Pharm.Pharmacol. (1997), 49:16-21; Lundberg, Anti-cancer Drug Design (1998),13: 453-461. See also U.S. Pat. No. 6,306,393; U.S. Ser. No. 10/350,096;U.S. Ser. No. 09/590,284, and U.S. Ser. No. 60/138,284, filed Jun. 9,1999. All these references are incorporated herein by reference.

A wide variety of diagnostic and therapeutic agents can beadvantageously used to form the conjugates of the stably tetheredstructures, or may be linked to haptens that bind to a recognition siteon the stably tethered structures. Diagnostic agents may includeradioisotopes, enhancing agents for use in MRI or contrast agents forultrasound imaging, and fluorescent compounds. Many appropriate imagingagents are known in the art, as are methods for their attachment toproteins or peptides (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509,both incorporated herein by reference). Certain attachment methodsinvolve the use of a metal chelate complex employing, for example, anorganic chelating agent such a DTPA attached to the protein or peptide(U.S. Pat. No. 4,472,509).

In order to load a stably tethered structure with radioactive metals orparamagnetic ions, it may be necessary to first react it with a carrierto which multiple copies of a chelating group for binding theradioactive metals or paramagnetic ions have been attached. Such acarrier can be a polylysine, polysaccharide, or a derivatized orderivatizable polymeric substance having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and thelike known to be useful for this purpose. Carriers containing chelatesare coupled to the stably tethered structure using standard chemistriesin a way to minimize aggregation and loss of immunoreactivity.

Other, more unusual, methods and reagents that may be applied forpreparing such conjugates are disclosed in U.S. Pat. No. 4,824,659,which is incorporated herein in its entirety by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV. Some useful diagnostic nuclidesmay include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc,^(94m)Tc, ^(99m)Tc, or ¹¹¹In. The same chelates complexed withnon-radioactive metals, such as manganese, iron and gadolinium, areuseful for MRI, when used along with the stably tethered structures andcarriers described herein. Macrocyclic chelates such as NOTA, DOTA, andTETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates, such as macrocyclic polyethers for complexing ²²³Ra, may beused.

Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and proapoptotic agents, particularlydoxorubicin, methotrexate, TAXOL®, CPT-11, camptothecans, and othersfrom these and other classes of anticancer agents, and the like. Othercancer chemotherapeutic drugs include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidineanalogs, purine analogs, platinum coordination complexes, hormones, andthe like. Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art, and may beconjugated to the stably tethered structures described herein usingmethods that are known in the art.

Another class of therapeutic agents consists of radionuclides that emitα-particles (such as ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac),β-particles (such as ³²P, ³³P, ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re), orAuger electrons (such as ¹¹¹In, ¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt,^(195m)Hg). The stably tethered structures may be labeled with one ormore of the above radionuclides using methods as described for thediagnostic agents.

A suitable peptide containing a detectable label (e.g., a fluorescentmolecule), or a cytotoxic agent, (e.g., a radioiodine), can becovalently, non-covalently, or otherwise associated with the stablytethered structures. For example, a therapeutically useful conjugate canbe obtained by incorporating a photoactive agent or dye onto the stablytethered structures. Fluorescent compositions, such as fluorochrome, andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. In therapy, this has been termedphotoradiation, phototherapy, or photodynamic therapy. See Jori et al.(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (LibreriaProgetto 1985); van den Bergh, Chem. Britain (1986), 22:430. Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. See Mew et al., J. Immunol. (1983), 130:1473;idem., Cancer Res. (1985), 45:4380; Oseroff et al., Proc. Natl. Acad.Sci. USA (1986), 83:8744; idem., Photochem. Photobiol. (1987), 46:83;Hasan et al., Prog. Clin. Biol. Res. (1989), 288:471; Tatsuta et al.,Lasers Surg. Med. (1989), 9:422; Pelegrin et al., Cancer (1991),67:2529. Endoscopic applications are also contemplated. Endoscopicmethods of detection and therapy are described in U.S. Pat. Nos.4,932,412; 5,525,338; 5,716,595; 5,736,119; 5,922,302; 6,096,289; andU.S. Pat. No. 6,387,350, which are incorporated herein by reference intheir entirety.

In certain embodiments, the novel constructs and methods disclosedherein are useful for targeted delivery of RNAi for therapeuticintervention. The delivery vehicle can be either an a2 (dimer) or an a4(tetramer) structure with an internalizing antibody binding domain fusedto human protamine (peptide of ˜50 amino acid residues) as itsprecursor. An example of an a2 construct would be VH—CH1-hP1-DDD1//VL-CLor VH—CH1-hP2-DDD1//VL-CL, where hP1 and hP2 are human protamine 1 andhuman protamine 2, respectively; both capable of forming stable DNAcomplexes for in vivo applications (Nat Biotechnol. 23: 709-717, 2005;Gene Therapy. 13: 194-195, 2006). An example of an a4 construct would beVH—CH1-hP1-DDD2//VL-CL or VH—CH1-hP2-DDD2//VL-CL, which would providefour active Fab fragments, each carrying a human protamine for bindingto RNAi. The multivalent complex will facilitate the binding to andreceptor-mediated internalization into target cells, where thenoncovalently bound RNAi is dissociated in the endosomes and releasedinto cytoplasm. As no redox chemistry is involved, the existence of 3intramolecular disulfide bonds in hP1 or hP2 does not present a problem.In addition to delivery of RNAi, these constructs may also be of use fortargeted delivery of therapeutic genes or DNA vaccines. Another area ofuse is to apply the A4/A2 technology for producing intrabodies, which isthe protein analog of RNAi in terms of function.

Peptide Administration

Various embodiments of the claimed methods and/or compositions mayconcern one or more peptide based stably tethered structures to beadministered to a subject. Administration may occur by any route knownin the art, including but not limited to oral, nasal, buccal,inhalational, rectal, vaginal, topical, orthotopic, intradermal,subcutaneous, intramuscular, intraperitoneal, intraarterial, intrathecalor intravenous injection.

Unmodified peptides administered orally to a subject can be degraded inthe digestive tract and depending on sequence and structure may exhibitpoor absorption across the intestinal lining. However, methods forchemically modifying peptides to render them less susceptible todegradation by endogenous proteases or more absorbable through thealimentary tract are well known (see, for example, Blondelle et al.,1995, Biophys. J. 69:604-11; Ecker and Crooke, 1995, Biotechnology13:351-69; Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMISTRY AND DRUGDISCOVERY, VOL. I, ed. Wollf, John Wiley & Sons; Goodman and Shao, 1996,Pure & Appl. Chem. 68:1303-08). Methods for preparing libraries ofpeptide analogs, such as peptides containing D-amino acids;peptidomimetics consisting of organic molecules that mimic the structureof a peptide; or peptoids such as vinylogous peptoids, have also beendescribed and may be used to construct peptide based stably tetheredstructures suitable for oral administration to a subject.

In certain embodiments, the standard peptide bond linkage may bereplaced by one or more alternative linking groups, such as CH₂—NH,CH₂—S, CH₂—CH₂, CH═CH, CO—CH₂, CHOH—CH₂ and the like. Methods forpreparing peptide mimetics are well known (for example, Hruby, 1982,Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest etal., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int. J. Pept.Res. 14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Pat.Nos. 5,169,862; 5,539,085; 5,576,423, 5,051,448, 5,559,103, eachincorporated herein by reference.) Peptide mimetics may exhibit enhancedstability and/or absorption in vivo compared to their peptide analogs.

Alternatively, peptides may be administered by oral delivery usingN-terminal and/or C-terminal capping to prevent exopeptidase activity.For example, the C-terminus may be capped using amide peptides and theN-terminus may be capped by acetylation of the peptide. Peptides mayalso be cyclized to block exopeptidases, for example by formation ofcyclic amides, disulfides, ethers, sulfides and the like.

Peptide stabilization may also occur by substitution of D-amino acidsfor naturally occurring L-amino acids, particularly at locations whereendopeptidases are known to act. Endopeptidase binding and cleavagesequences are known in the art and methods for making and using peptidesincorporating D-amino acids have been described (e.g., U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Jun. 14,2004, incorporated herein by reference). In certain embodiments,peptides and/or proteins may be orally administered by co-formulationwith proteinase- and/or peptidase-inhibitors.

Other methods for oral delivery of therapeutic peptides are disclosed inMehta (“Oral delivery and recombinant production of peptide hormones,”June 2004, BioPharm International). The peptides are administered in anenteric-coated solid dosage form with excipients that modulateintestinal proteolytic activity and enhance peptide transport across theintestinal wall. Relative bioavailability of intact peptides using thistechnique ranged from 1% to 10% of the administered dosage. Insulin hasbeen successfully administered in dogs using enteric-coatedmicrocapsules with sodium cholate and a protease inhibitor (Ziv et al.,1994, J. Bone Miner. Res. 18 (Suppl. 2):792-94. Oral administration ofpeptides has been performed using acylcarnitine as a permeation enhancerand an enteric coating (Eudragit L30D-55, Rohm Pharma Polymers, seeMehta, 2004). Excipients of use for orally administered peptides maygenerally include one or more inhibitors of intestinalproteases/peptidases along with detergents or other agents to improvesolubility or absorption of the peptide, which may be packaged within anenteric-coated capsule or tablet (Mehta, 2004). Organic acids may beincluded in the capsule to acidify the intestine and inhibit intestinalprotease activity once the capsule dissolves in the intestine (Mehta,2004). Another alternative for oral delivery of peptides would includeconjugation to polyethylene glycol (PEG)-based amphiphilic oligomers,increasing absorption and resistance to enzymatic degradation (Solteroand Ekwuribe, 2001, Pharm. Technol. 6:110).

In still other embodiments, peptides may be modified for oral orinhalational administration by conjugation to certain proteins, such asthe Fc region of IgG1 (see Examples 3-7). Methods for preparation anduse of peptide-Fc conjugates are disclosed, for example, in Low et al.(2005, Hum. Reprod. 20:1805-13) and Dumont et al. (2005, J. Aerosol.Med. 18:294-303), each incorporated herein by reference. Low et al.(2005) disclose the conjugation of the alpha and beta subunits of FSH tothe Fc region of IgG1 in single chain or heterodimer form, usingrecombinant expression in CHO cells. The Fc conjugated peptides wereabsorbed through epithelial cells in the lung or intestine by theneonatal Fc receptor mediated transport system. The Fc conjugatedpeptides exhibited improved stability and absorption in vivo compared tothe native peptides. It was also observed that the heterodimer conjugatewas more active than the single chain form.

Proteins and Peptides

A variety of polypeptides or proteins may be used within the scope ofthe claimed methods and compositions. In certain embodiments, theproteins may comprise antibodies or fragments of antibodies containingan antigen-binding site. As used herein, a protein, polypeptide orpeptide generally refers, but is not limited to, a protein of greaterthan about 200 amino acids, up to a full length sequence translated froma gene; a polypeptide of greater than about 100 amino acids; and/or apeptide of from about 3 to about 100 amino acids. For convenience, theterms “protein,” “polypeptide” and “peptide” are used interchangeablyherein. Accordingly, the term “protein or peptide” encompasses aminoacid sequences comprising at least one of the 20 common amino acidsfound in naturally occurring proteins, or at least one modified orunusual amino acid.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to those shown below.

Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3-Aminoadipic acid HylHydroxylysine Bala β-alanine, β-Amino- AHyl allo-Hydroxylysine propionicacid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyricacid, 4Hyp 4-Hydroxyproline piperidinic acid Acp 6-Aminocaproic acid IdeIsodesmosine Ahe 2-Aminoheptanoic acid AIle allo-Isoleucine Aib2-Aminoisobutyric acid MeGly N-Methylglycine, sarcosine Baib3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm 2-Aminopimelic acidMeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acid MeVal N-MethylvalineDes Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelic acid Nle NorleucineDpr 2,3-Diaminopropionic acid Orn Ornithine EtGly N-Ethylglycine

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases(www.ncbi.nlm.nih.gov/). The coding regions for known genes may beamplified and/or expressed using the techniques disclosed herein or aswould be know to those of ordinary skill in the art. Alternatively,various commercial preparations of proteins, polypeptides, and peptidesare known to those of skill in the art.

Peptide Mimetics

Another embodiment for the preparation of polypeptides is the use ofpeptide mimetics. Mimetics are peptide-containing molecules that mimicelements of protein secondary structure. See, for example, Johnson etal., “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto etal., Eds., Chapman and Hall, New York (1993), incorporated herein byreference. The rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains so as to facilitate molecular interactions, such as those ofantibody and antigen. A peptide mimetic is expected to permit molecularinteractions similar to the natural molecule.

Fusion Proteins

Various embodiments may concern fusion proteins. These moleculesgenerally have all or a substantial portion of a peptide, linked at theN- or C-terminus, to all or a portion of a second polypeptide orprotein. Methods of generating fusion proteins are well known to thoseof skill in the art. Such proteins may be produced, for example, bychemical attachment using bifunctional cross-linking reagents, by denovo synthesis of the complete fusion protein, or by attachment of a DNAsequence encoding a first protein or peptide to a DNA sequence encodinga second peptide or protein, followed by expression of the intact fusionprotein.

Synthetic Peptides

Proteins or peptides may be synthesized, in whole or in part, insolution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, (1984, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co.); Tam et al., (1983, J. Am. Chem. Soc., 105:6442);Merrifield, (1986, Science, 232: 341-347); and Barany and Merrifield(1979, The Peptides, Gross and Meienhofer, eds., Academic Press, NewYork, pp. 1-284). Short peptide sequences, usually from about 6 up toabout 35 to 50 amino acids, can be readily synthesized by such methods.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of interest is inserted intoan expression vector, transformed or transfected into an appropriatehost cell, and cultivated under conditions suitable for expression.

Antibodies

Various embodiments may concern antibodies for a target. The term“antibody” is used herein to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlowe and Lane, 1988, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory). Antibodies of use may also becommercially obtained from a wide variety of known sources. For example,a variety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.).

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments may be produced by enzymatic cleavage ofantibodies with pepsin to provide F(ab′)₂ fragments. This fragment maybe further cleaved using a thiol reducing agent and, optionally,followed by a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using papain n produces twomonovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. No. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFv's are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.).

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immunol., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990).

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods, as known in the art. The skilled artisanwill realize that this technique is exemplary only and any known methodfor making and screening human antibodies or antibody fragments by phagedisplay may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled to other molecules bychemical cross-linking or other known methodologies. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXenoMouse® system but may utilize any transgenic animal that has beengenetically engineered to produce human antibodies.

Pre-Targeting

One strategy for use of bi-specific stably tethered constructs includespre-targeting methodologies, in which an effector molecule isadministered to a subject after a bi-specific construct has beenadministered. The bi-specific construct, which would include a bindingsite for an effector, hapten or carrier and one for the diseased tissue,localizes to the diseased tissue and increases the specificity oflocalization of the effector to the diseased tissue (U.S. PatentApplication No. 20050002945). Because the effector molecule may becleared from circulation much more rapidly than the bi-specificconstruct, normal tissues may have a decreased exposure to the effectormolecule when a pre-targeting strategy is used than when the effectormolecule is directly linked to the disease targeting antibody.

Pre-targeting methods have been developed to increase thetarget:background ratios of detection or therapeutic agents. Examples ofpre-targeting and biotin/avidin approaches are described, for example,in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl.Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehret al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. Nos. 6,077,499; 09/597,580; 10/361,026; 09/337,756;09/823,746; 10/116,116; 09/382,186; 10/150,654; 6,090,381; 6,472,511;10/114,315; U.S. Provisional Application No. 60/386,411; U.S.Provisional Application No. 60/345,641; U.S. Provisional Application No.60/3328,835; U.S. Provisional Application No. 60/426,379; U.S. Ser. Nos.09/823,746; 09/337,756; and U.S. Provisional Application No. 60/342,103,all of which are incorporated herein by reference.

In certain embodiments, bi-specific constructs and targetable constructsmay be of use in treating and/or imaging normal or diseased tissue andorgans, for example using the methods described in U.S. Pat. Nos.6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093;5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119;5,101,827; and 4,735,210, each incorporated herein by reference.Additional methods are described in U.S. application Ser. No. 09/337,756filed Jun. 22, 1999 and in U.S. application Ser. No. 09/823,746, filedApr. 3, 2001.

Aptamers

In certain embodiments, a precursor for construct formation may comprisean aptamer. Methods of constructing and determining the bindingcharacteristics of aptamers are well known in the art. For example, suchtechniques are described in U.S. Pat. Nos. 5,582,981, 5,595,877 and5,637,459, each incorporated herein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the binding sequences of aptamers may be flankedby primer-binding sequences, facilitating the amplification of theaptamers by PCR or other amplification techniques. In a furtherembodiment, the flanking sequence may comprise a specific sequence thatpreferentially recognizes or binds a moiety to enhance theimmobilization of the aptamer to a substrate.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Methods for preparation and screening of aptamers that bind toparticular targets of interest are well known, for example U.S. Pat. No.5,475,096 and U.S. Pat. No. 5,270,163, each incorporated by reference.The technique generally involves selection from a mixture of candidateaptamers and step-wise iterations of binding, separation of bound fromunbound aptamers and amplification. Because only a small number ofsequences (possibly only one molecule of aptamer) corresponding to thehighest affinity aptamers exist in the mixture, it is generallydesirable to set the partitioning criteria so that a significant amountof aptamers in the mixture (approximately 5-50%) is retained duringseparation. Each cycle results in an enrichment of aptamers with highaffinity for the target. Repetition for between three to six selectionand amplification cycles may be used to generate aptamers that bind withhigh affinity and specificity to the target.

Avimers

In certain embodiments, the precursors, components and/or complexesdescribed herein may comprise one or more avimer sequences. Avimers area class of binding proteins somewhat similar to antibodies in theiraffinities and specificities for various target molecules. They weredeveloped from human extracellular receptor domains by in vitro exonshuffling and phage display. (Silverman et al., 2005, Nat. Biotechnol.23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220.) Theresulting multidomain proteins may comprise multiple independent bindingdomains that may exhibit improved affinity (in some cases sub-nanomolar)and specificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, DDDsequences for use in the claimed methods and compositions. Additionaldetails concerning methods of construction and use of avimers aredisclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Methods of Disease Tissue Detection, Diagnosis and Imaging

Protein-Based In Vitro Diagnosis

The present invention contemplates the use of stably tethered structuresto screen biological samples in vitro and/or in vivo for the presence ofthe disease-associated antigens. In exemplary immunoassays, a stablytethered structure comprising an antibody, fusion protein, or fragmentthereof may be utilized in liquid phase or bound to a solid-phasecarrier, as described below. In preferred embodiments, particularlythose involving in vivo administration, the antibody or fragment thereofis humanized. Also preferred, the antibody or fragment thereof is fullyhuman. Still more preferred, the fusion protein comprises a humanized orfully human antibody. The skilled artisan will realize that a widevariety of techniques are known for determining levels of expression ofa particular gene and any such known method, such as immunoassay,RT-PCR, mRNA purification and/or cDNA preparation followed byhybridization to a gene expression assay chip may be utilized todetermine levels of expression in individual subjects and/or tissues.Exemplary in vitro assays of use include RIA, ELISA, sandwich ELISA,Western blot, slot blot, dot blot, and the like. Although suchtechniques were developed using intact antibodies, stably tetheredstructures that incorporate antibodies, antibody fragments or otherbinding moieties may be used.

Stably tethered structures incorporating antibodies, fusion proteins,antibody fragments and/or other binding moieties may also be used todetect the presence of a target antigen in tissue sections prepared froma histological specimen. Such in situ detection can be used to determinethe presence of the antigen and to determine the distribution of theantigen in the examined tissue. In situ detection can be accomplished byapplying a detectably-labeled structure to frozen or paraffin-embeddedtissue sections. General techniques of in situ detection are well-knownto those of ordinary skill. See, for example, Ponder, “Cell MarkingTechniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICALAPPROACH 113-38 Monk (ed.) (IRL Press 1987), and Coligan at pages5.8.1-5.8.8.

Stably tethered structures can be detectably labeled with anyappropriate marker moiety, for example, a radioisotope, an enzyme, afluorescent label, a dye, a chromogen, a chemiluminescent label, abioluminescent label or a paramagnetic label.

The marker moiety may be a radioisotope that is detected by such meansas the use of a gamma counter or a beta-scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety refersto a molecule that will generate a signal under predeterminedconditions. Examples of marker moieties include radioisotopes, enzymes,fluorescent labels, chemiluminescent labels, bioluminescent labels andparamagnetic labels. The binding of marker moieties to stably tetheredstructures can be accomplished using standard techniques known to theart. Typical methodology in this regard is described by Kennedy et al.,Clin. Chim. Acta 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1(1977), Shih et al., Int'l J. Cancer 46: 1101 (1990).

Nucleic Acid Based In Vitro Diagnosis

Stably tethered structures may, in some embodiments, incorporatednucleic acid moieties. In particular embodiments, nucleic acids may beanalyzed to determine levels of binding, particularly using nucleic acidamplification methods. Various forms of amplification are well known inthe art and any such known method may be used. Generally, amplificationinvolves the use of one or more primers that hybridize selectively orspecifically to a target nucleic acid sequence to be amplified.

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Computerized programs for selection anddesign of amplification primers are available from commercial and/orpublic sources well known to the skilled artisan. A number of templatedependent processes are available to amplify the marker sequencespresent in a given sample. One of the best-known amplification methodsis the polymerase chain reaction (referred to as PCR), which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159. However, other methods of amplification are known and may beused.

In Vivo Diagnosis

Methods of diagnostic imaging with labeled peptides or MAbs arewell-known. For example, in the technique of immunoscintigraphy, ligandsor antibodies are labeled with a gamma-emitting radioisotope andintroduced into a patient. A gamma camera is used to detect the locationand distribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Alsopreferred is the use of positron-emitting radionuclides (PET isotopes),such as with an energy of 511 keV, such as ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, and ¹²⁴I.Such imaging can be conducted by direct labeling of the stably tetheredstructure, or by a pretargeted imaging method, as described inGoldenberg et al, “Antibody Pre-targeting Advances CancerRadioimmunodetection and Radioimmunotherapy,” (J Clin Oncol 2006;24:823-834), see also U.S. Patent Publication Nos. 20050002945,20040018557, 20030148409 and 20050014207, each incorporated herein byreference.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that are appropriate fordiagnostic imaging include ^(99m)Tc and ¹¹¹In.

The stably tethered structures, or haptens or carriers that bind tothem, also can be labeled with paramagnetic ions and a variety ofradiological contrast agents for purposes of in vivo diagnosis. Contrastagents that are particularly useful for magnetic resonance imagingcomprise gadolinium, manganese, dysprosium, lanthanum, or iron ions.Additional agents include chromium, copper, cobalt, nickel, rhenium,europium, terbium, holmium, or neodymium. ligands, antibodies andfragments thereof can also be conjugated to ultrasoundcontrast/enhancing agents. For example, one ultrasound contrast agent isa liposome that comprises a humanized IgG or fragment thereof. Alsopreferred, the ultrasound contrast agent is a liposome that is gasfilled.

Imaging Agents and Radioisotopes

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to proteins or peptides (see, e.g., U.S. Pat. Nos.5,021,236 and 4,472,509, both incorporated herein by reference). Certainattachment methods involve the use of a metal chelate complex employing,for example, an organic chelating agent such a DTPA attached to theprotein or peptide (U.S. Pat. No. 4,472,509). Proteins or peptides alsomay be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, carbon¹⁴, chromium⁵¹, chlorine³⁶, cobalt⁵⁷, cobalt⁵⁸,copper⁶², copper⁶⁴, copper⁶⁷, Eu¹⁵², fluorine¹⁸, gallium⁶⁷, gallium⁶⁸,hydrogen³, iodine¹²³, iodine¹²⁴, iodine¹²⁵, iodine¹³¹, indium¹¹¹,iron⁵², iron⁵⁹, lutetium¹⁷⁷, phosphorus32, phosphorus³³, rhenium¹⁸⁶,rhenium¹⁸⁸, Sc⁴⁷, selenium⁷⁵, silver¹¹¹, sulphur³⁵, technetium^(94m),technetium^(99m), yttrium⁸⁶ yttrium⁹⁰, and zirconium⁸⁹. I¹²⁵ is oftenbeing preferred for use in certain embodiments, and technetium^(99m) andindium¹¹¹ are also often preferred due to their low energy andsuitability for long-range detection.

Radioactively labeled proteins or peptides may be produced according towell-known methods in the art. For instance, they can be iodinated bycontact with sodium or potassium iodide and a chemical oxidizing agentsuch as sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Proteins or peptides may be labeled withtechnetium-^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the peptide. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto peptides include diethylenetriaminepentaacetic acid (DTPA), DOTA,NOTA, porphyrin chelators and ethylene diaminetetracetic acid (EDTA).Also contemplated for use are fluorescent labels, including rhodamine,fluorescein isothiocyanate and renographin.

In certain embodiments, the proteins or peptides may be linked to asecondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Preferredsecondary binding ligands are biotin and avidin or streptavidincompounds. The use of such labels is well known to those of skill in theart in light and is described, for example, in U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241;each incorporated herein by reference. These fluorescent labels arepreferred for in vitro uses, but may also be of utility in in vivoapplications, particularly endoscopic or intravascular detectionprocedures.

In alternative embodiments, ligands, antibodies, or other proteins orpeptides may be tagged with a fluorescent marker. Non-limiting examplesof photodetectable labels include Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole),Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, phthalocyanines, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, Edans and Texas Red. These and other luminescentlabels may be obtained from commercial sources such as Molecular Probes(Eugene, Oreg.), and EMD Biosciences (San Diego, Calif.).

Chemiluminescent labeling compounds of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt and an oxalate ester, or a bioluminescent compound such asluciferin, luciferase and aequorin. Diagnostic conjugates may be used,for example, in intraoperative, endoscopic, or intravascular tumor ordisease diagnosis.

In various embodiments, labels of use may comprise metal nanoparticles.Methods of preparing nanoparticles are known. (See e.g., U.S. Pat. Nos.6,054,495; 6,127,120; 6,149,868; Lee and Meisel, J. Phys. Chem.86:3391-3395, 1982.) Nanoparticles may also be obtained from commercialsources (e.g., Nanoprobes Inc., Yaphank, N.Y.; Polysciences, Inc.,Warrington, Pa.). Modified nanoparticles are available commercially,such as Nanogold® nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.).Functionalized nanoparticles of use for conjugation to proteins orpeptides may be commercially obtained.

Therapeutic Agents

Pharmaceutical Compositions

In some embodiments, a stably tethered structure and/or one or moreother therapeutic agents may be administered to a subject, such as asubject with cancer. Such agents may be administered in the form ofpharmaceutical compositions. Generally, this will entail preparingcompositions that are essentially free of impurities that could beharmful to humans or animals. One skilled in the art would know that apharmaceutical composition can be administered to a subject by variousroutes including, for example, orally or parenterally, such asintravenously.

In certain embodiments, an effective amount of a therapeutic agent mustbe administered to the subject. An “effective amount” is the amount ofthe agent that produces a desired effect. An effective amount willdepend, for example, on the efficacy of the agent and on the intendedeffect. For example, a lesser amount of an antiangiogenic agent may berequired for treatment of a hyperplastic condition, such as maculardegeneration or endometriosis, compared to the amount required forcancer therapy in order to reduce or eliminate a solid tumor, or toprevent or reduce its metastasizing. An effective amount of a particularagent for a specific purpose can be determined using methods well knownto those in the art.

Chemotherapeutic Agents

In certain embodiments, chemotherapeutic agents may be administered.Anti-cancer chemotherapeutic agents of use include, but are not limitedto, 5-fluorouracil, bleomycin, busulfan, camptothecins, carboplatin,chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin,daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide(VP16), farnesyl-protein transferase inhibitors, gemcitabine,ifosfamide, mechlorethamine, melphalan, methotrexate, mitomycin,navelbine, nitrosurea, plicamycin, procarbazine, raloxifene, tamoxifen,TAXOL®, temazolomide (an aqueous form of DTIC), transplatinum,vinblastine and methotrexate, vincristine, or any analog or derivativevariant of the foregoing. Chemotherapeutic agents of use againstinfectious organisms include, but are not limited to, acyclovir,albendazole, amantadine, amikacin, amoxicillin, amphotericin B,ampicillin, aztreonam, azithromycin, bacitracin, BACTRIM®, Batrafen®,bifonazole, carbenicillin, caspofungin, cefaclor, cefazolin,cephalosporins, cefepime,ceftriaxone, cefotaxime, chloramphenicol,cidofovir, Cipro®, clarithromycin, clavulanic acid, clotrimazole,cloxacillin, doxycycline, econazole, erythrocycline, erythromycin,FLAGYL®, fluconazole, flucytosine, FOSCARNET®, furazolidone,ganciclovir, gentamycin, imipenem, isoniazid, itraconazole, kanamycin,ketoconazole, lincomycin, linezolid, meropenem, miconazole, minocycline,naftifine, nalidixic acid, neomycin, netilmicin, nitrofurantoin,nystatin, oseltamivir, oxacillin, paromomycin, penicillin, pentamidine,piperacillin-tazobactam, rifabutin, rifampin, rimantadine, streptomycin,sulfamethoxazole, sulfasalazine, tetracycline, tioconazole, tobramycin,tolciclate, tolnaftate, trimethoprim sulfamethoxazole, valacyclovir,vancomycin, zanamir, and zithromycin.

Chemotherapeutic agents and methods of administration, dosages, etc.,are well known to those of skill in the art (see for example, the“Physicians Desk Reference”, Goodman & Gilman's “The PharmacologicalBasis of Therapeutics” and in “Remington's Pharmaceutical Sciences”,incorporated herein by reference in relevant parts). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.

Hormones

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones. Progestins, such as hydroxyprogesteronecaproate, medroxyprogesterone acetate, and megestrol acetate, have beenused in cancers of the endometrium and breast. Estrogens such asdiethylstilbestrol and ethinyl estradiol have been used in cancers suchas prostate cancer. Antiestrogens such as tamoxifen have been used incancers such as breast cancer. Androgens such as testosterone propionateand fluoxymesterone have also been used in treating breast cancer.

Angiogenesis Inhibitors

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PIGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, lamininpeptides, fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16Kprolactin fragment, Linomide, thalidomide, pentoxifylline, genistein,TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline maybe of use.

Immunomodulators

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins and hematopoietic factors, such asinterleukins, colony-stimulating factors, interferons (e.g.,interferons-α, -β and -γ) and the stem cell growth factor designated “S1factor.” Examples of suitable immunomodulator moieties include IL-2,IL-6, IL-10, IL-12, IL-18, IL-21, interferon-gamma, TNF-alpha, and thelike.

The term “cytokine” is a generic term for proteins or peptides releasedby one cell population which act on another cell as intercellularmediators. As used broadly herein, examples of cytokines includelymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; prostaglandin, fibroblast growth factor;prolactin; placental lactogen, OB protein; tumor necrosis factor-α and-β; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, Il-15, IL-16,IL-17, IL-18, IL-21, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. Chemokines include, but arenot limited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. Theskilled artisan will recognize that certain cytokines are also known tohave chemoattractant effects and could also be classified under the termchemokines. Similarly, the terms immunomodulator and cytokine overlap intheir respective members.

Radioisotope Therapy and Radioimmunotherapy

In some embodiments, the peptides and/or proteins may be of use inradionuclide therapy or radioimmunotherapy methods (see, e.g., Govindanet al., 2005, Technology in Cancer Research & Treatment, 4:375-91;Sharkey and Goldenberg, 2005, J. Nucl. Med. 46:115S-127S; Goldenberg etal. (J Clin Oncol 2006; 24:823-834), “Antibody Pre-targeting AdvancesCancer Radioimmunodetection and Radioimmunotherapy,” each incorporatedherein by reference.) In specific embodiments, stably tetheredstructures may be directly tagged with a radioisotope of use andadministered to a subject. In alternative embodiments, radioisotope(s)may be administered in a pre-targeting method as discussed above, usinga haptenic peptide or ligand that is radiolabeled and injected afteradministration of a bispecific stably tethered structure that localizesat the site of elevated expression in the diseased tissue.

Radioactive isotopes useful for treating diseased tissue include, butare not limited to-¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.The therapeutic radionuclide preferably has a decay energy in the rangeof 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Augeremitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for analpha emitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5 hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to a proteinor peptide using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA).Alternatively, ⁹⁰Y, which emits an energetic beta particle, can becoupled to a peptide, antibody, fusion protein, or fragment thereof,using diethylenetriaminepentaacetic acid (DTPA).

Additional potential radioisotopes include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au,²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used. The addition ofthe radiosensitizer can result in enhanced efficacy. Radiosensitizersare described in D. M. Goldenberg (ed.), CANCER THERAPY WITHRADIOLABELED ANTIBODIES, CRC Press (1995), which is incorporated hereinby reference in its entirety.

The peptide, antibody, antibody fragment, or fusion protein that has aboron addend-loaded carrier for thermal neutron activation therapy willnormally be effected in similar ways. However, it will be advantageousto wait until non-targeted immunoconjugate clears before neutronirradiation is performed. Clearance can be accelerated using an antibodythat binds to the ligand. See U.S. Pat. No. 4,624,846 for a descriptionof this general principle. For example, boron addends such ascarboranes, can be attached to antibodies. Carboranes can be preparedwith carboxyl functions on pendant side chains, as is well-known in theart. Attachment of carboranes to a carrier, such as aminodextran, can beachieved by activation of the carboxyl groups of the carboranes andcondensation with amines on the carrier. The intermediate conjugate isthen conjugated to the antibody. After administration of the conjugate,a boron addend is activated by thermal neutron irradiation and convertedto radioactive atoms which decay by alpha-emission to produce highlytoxic, short-range effects.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one stably tethered structure. If the compositioncontaining components for administration is not formulated for deliveryvia the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used.

The kit components may be packaged together or separated into two ormore separate containers. In some embodiments, the containers may bevials that contain sterile, lyophilized formulations of a compositionthat are suitable for reconstitution. A kit may also contain one or morebuffers suitable for reconstitution and/or dilution of other reagents.Other containers that may be used include, but are not limited to, apouch, tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The following examples are provided to illustrate, but not to limit theclaimed invention.

Example 1 General Strategy for Producing Fab-Based Subunits with theDDD1 Sequence Appended to Either the C- or N-Terminus of the Fd Chain

Fab-based subunits with the DDD1 sequence (SEQ ID NO:1) appended toeither the C- or N-terminus of the Fd chain are produced as fusionproteins. The plasmid vector pdHL2 has been used to produce a number ofantibodies and antibody-based constructs. See Gillies et al., J ImmunolMethods (1989), 125:191-202; Losman et al., Cancer (Phila) (1997),80:2660-6. The di-cistronic mammalian expression vector directs thesynthesis of the heavy and light chains of IgG. The vector sequences aremostly identical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, theseIgG-pdHL2 expression vectors can be converted into Fd-DDD1-pdHL2 orFd-DDD2-pdHL2 expression vectors by replacing the coding sequences forthe hinge, CH2 and CH3 domains of the heavy chain with a sequenceencoding the first 4 residues of the hinge, a 14 residue Gly-Ser linkerand the first 44 residues of human RIIα. The shuttle vectorCH1-DDD1-pGemT was designed to facilitate the conversion of IgG-pdHL2vectors (FIG. 2 a) to Fd-DDD1-pdHL2 vectors (FIG. 2 b), as describedbelow.

Generation of the Shuttle Vector CH1-DDD1-pGemT

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consists of the upstream (5′) of the CH1domain and a SacII restriction endonuclease site, which is 5′ of the CH1coding sequence. The right primer consists of the sequence coding forthe first 4 residues of the hinge (PKSC) followed by GGGGS with thefinal two codons (GS) comprising a Bam HI restriction site.

5′ of CH1 Left Primer (SEQ ID NO: 4) 5′GAACCTCGCGGACAGTTAAG-3′ CH1 +G₄S-Bam Right (SEQ ID NO: 5)5′GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTG GTGTTGCTGG-3′

The 410 bp PCR amplimer was cloned into the pGemT PCR cloning vector(Promega, Inc.) and clones were screened for inserts in the T7 (5′)orientation.

Construction of (G₄S)₂DDD1

A duplex oligonucleotide, designated (G₄S)₂DDD1, was synthesized bySigma Genosys (Haverhill, UK) to code for the amino acid sequence ofDDD1 (SEQ ID NO:1) preceded by 11 residues of the linker peptide, withthe first two codons comprising a BamHI restriction site. A stop codonand an EagI restriction site are appended to the 3′end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 6) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFT RLREARA

The two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,which overlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174bp DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase.

RIIA1-44 top (SEQ ID NO: 7) 5′GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAG GTGCTGCGACAG-3′RIIA1-44 bottom (SEQ ID NO: 8)5′GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGT AGCCCTG-3′

Following primer extension, the duplex was amplified by PCR using thefollowing primers:

G4S Bam-Left (SEQ ID NO: 9) 5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′1-44 stop Eag Right (SEQ ID NO: 10) 5′-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3′

This amplimer was cloned into pGemT and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

Cloning CH1-DDD1 into pdHL2-Based Vectors

The sequence encoding CH1-DDD1 can be incorporated into any IgGconstruct in the pdHL2 vector as follows. The entire heavy chainconstant domain is replaced with CH1-DDD1 by removing the SacII/EagIrestriction fragment (CH1-CH3) from pdHL2 and replacing it with theSacII/EagI fragment of CH1-DDD1, which is excised from the respectivepGemT shuttle vector.

It is noted that the location of DDD1 is not restricted to the carboxylterminal end of CH1 and can be placed at the amino terminal end of theVH domain, as shown in Example 2.

Example 2 Methods for Generating a₂ Constructs Composed of Two IdenticalFab Subunits Stably Linked Via the DDD1 Sequence Fused to Either the C-or N-Terminus of the Fd Chain

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for producing an a₂construct that comprises two copies of a fusion protein in which theDDD1 sequence is linked to hMN-14 Fab at the C-terminus of the Fd chainvia a flexible peptide spacer (FIG. 3). The plasmid vectorhMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was convertedto C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restrictionendonucleases, to remove the fragment encoding the CH1-CH3 domains, andinsertion of the CH1-DDD1 fragment, which was excised from theCH1-DDD1-SV3 shuttle vector with SacII and EagI.

Construction of N-DDD1-Fd-hMN-14-pdHL2

N-DDD1-Fd-hMN-14-pdHL2 is an expression vector for producing an a₂construct that comprises two copies of a fusion protein in which theDDD1 sequence is linked to hMN-14 Fab at the N-terminus of the Fd chainvia a flexible peptide spacer (FIG. 4).

The expression vector was engineered as follows. The DDD1 domain wasamplified by PCR using the two primers shown below.

DDD1 Nco Left (SEQ ID NO: 11) 5′ CCATGGGCAGCCACATCCAGATCCCGCC-3′DDD1-G₄S Bam Right (SEQ ID NO: 12)5′GGATCCGCCACCTCCAGATCCTCCGCCGCCAGCGCGAGCTTCTCTCAG GCGGGTG-3′

As a result of the PCR, an NcoI restriction site and the coding sequencefor part of the linker (G₄S)₂ containing a BamHI restriction wereappended to the 5′ and 3′ ends, respectively. The 170 bp PCR amplimerwas cloned into the pGemT vector and clones were screened for inserts inthe T7 (5′) orientation. The 194 bp insert was excised from the pGemTvector with NcoI and SalI restriction enzymes and cloned into the SV3shuttle vector, which was prepared by digestion with those same enzymes,to generate the intermediate vector DDD1-SV3.

The hMN-14 Fd sequence was amplified by PCR using the oligonucleotideprimers shown below.

hMN-14VH left G4S Bam (SEQ ID NO: 13)5′-GGATCCGGCGGAGGTGGCTCTGAGGTCCAACTGGTGGAGAGCGG-3′ CH1-C stop Eag(SEQ ID NO: 14) 5′-CGGCCGTCAGCAGCTCTTAGGTTTCTTGTC-3′

As a result of the PCR, a BamHI restriction site and the coding sequencefor part of the linker (G₄S) were appended to the 5′ end of theamplimer. A stop codon and EagI restriction site was appended to the3′end. The 1043 bp amplimer was cloned into pGemT. The hMN-14-Fd insertwas excised from pGemT with BamHI and EagI restriction enzymes and thenligated with DDD1-SV3 vector, which was prepared by digestion with thosesame enzymes, to generate the construct N-DDD1-Fd-hMN-14-SV3.

The N-DDD1-hMN-14 Fd sequence was excised with XhoI and EagI restrictionenzymes and the 1.28 kb insert fragment was ligated with a vectorfragment that was prepared by digestion of C-DDD1-Fd-hMN-14-pdHL2 withthose same enzymes. The final expression vector isN-DDD1-Fd-hMN-14-pDHL2.

Production, Purification and Characterization of N-DDD1-Fab-hMN-14 andC-DDD1-Fab-hMN-14

The C-DDD1-Fd-hMN-14-pdHL2 and N-DDD1-Fd-hMN-14-pdHL2 vectors weretransfected into Sp2/0-derived myeloma cells by electroporation.C-DDD1-Fd-hMN-14-pdHL2 is a di-cistronic expression vector, whichdirects the synthesis and secretion of both hMN-14 kappa light chain andhMN-14 Fd-DDD1, which combine to form C-DDD1-hMN-14 Fab.N-DDD1-hMN-14-pdHL2 is a di-cistronic expression vector, which directsthe synthesis and secretion of both hMN-14 kappa light chain andN-DDD1-Fd-hMN-14, which combine to form N-DDD1-Fab-hMN-14. Each fusionprotein forms a stable homodimer via the interaction of the DDD1 domain.

Following electroporation, the cells were plated in 96-well tissueculture plates and transfectant clones were selected with 0.05 μMmethotrexate (MTX). Clones were screened for protein expression byELISA, using microtitre plates coated with WI2 (a rat anti-id monoclonalantibody to hMN-14) and detection with HRP-conjugated goat anti-humanFab. The initial productivity of the highest producing C-DDD1-Fab-hMN14and N-DDD1-Fab-hMN14 clones was 60 mg/L and 6 mg/L, respectively.

Both fusion proteins are purified using affinity chromatography. AD1-Cis a peptide that binds specifically to DDD1-containing a₂ constructs.The amino acid sequence of AD1-C (SEQ ID NO:3) is shown in FIG. 5. AD1-Cwas coupled to Affigel following reaction of the suithydryl group withchloroacetic anhydride. Culture supernatants were concentratedapproximately 10-fold by ultrafiltration before loading onto anAD1-C-affigel column. The column was washed to baseline with PBS andC-DDD1-Fab-hMN-14 was eluted with 0.1 M Glycine, pH 2.5. The one-stepaffinity purification yielded about 81 mg of C-DDD1-Fab-hMN-14 from 1.2liters of roller bottle culture. SE-HPLC analysis (FIG. 6) of the eluateshows a single protein peak with a retention time (8.7 min) consistentwith a 107-kDa protein. The purity was also confirmed by reducingSDS-PAGE (FIG. 7), showing only two bands of molecular size expected forthe two polypeptide constituents of C-DDD1-Fab-hMN-14.

N-DDD1-Fab-hMN-14 was purified as described above for C-DDD1-Fab-hMN-14,yielding 10 mg from 1.2 liters of roller bottle culture. SE-HPLCanalysis (FIG. 8) of the eluate shows a single protein peak with aretention time (8.77 min) similar to C-DDD1-Fab-hMN-14 and consistentwith a 107 kDa protein. Reducing SDS-PAGE shows only two bandsattributed to the polypeptide constituents of N-DDD1-Fab-hMN-14.

The binding activity of C-DDD1-Fab-hMN-14 was determined by SE-HPLCanalysis of samples in which the test article was mixed with variousamounts of WI2. A sample prepared by mixing WI2 Fab andC-DDD1-Fab-hMN-14 at a molar ratio of 0.75:1 showed three peaks, whichwere attributed to unbound C-DDD1-Fab-hMN14 (8.71 min.),C-DDD1-Fab-hMN-14 bound to one WI2 Fab (7.95 min.), and C-DDD1-Fab-hMN14bound to two WI2 Fabs (7.37 min.). When a sample containing WI2 Fab andC-DDD1-Fab-hMN-14 at a molar ratio of 4 was analyzed, only a single peakat 7.36 minutes was observed. These results (FIG. 9) demonstrate thatC-DDD1-Fab-hMN-14 is dimeric and has two active binding sites. Verysimilar results (FIG. 10) were obtained when this experiment wasrepeated with N-DDD1-Fab-hMN-14.

Competitive ELISA (FIGS. 11 and 12) demonstrated that C-DDD1-Fab-hMN-14and N-DDD1-Fab-hMN-14 binds to CEA with similar avidity to hMN-14 IgG,and significantly stronger than monovalent hMN-14 Fab. ELISA plates werecoated with a fusion protein containing the epitope (A3B3) of CEA towhich hMN-14 binds specifically. C-DDD1-Fab-hMN-14 is stable in pooledhuman serum for at least 24 h without appreciable loss inimmunoreactivity as shown in FIGS. 13 and 14. C-DDD1-Fab-hMN-14 has beenevaluated in mice bearing human colorectal cancer xenografts (LS174T)and the results (FIGS. 15 and 16) were similar to those obtained forhBS14-1, which is also bivalent for binding to CEA.

Example 3 Methods for Generating a₂ Constructs Composed of Two IdenticalFab Fusion Proteins, each Containing Ranpirnase (Rap) and the DDD1Sequence Linked to the N-Terminus of the Light Chain and the C-terminusof the Fd Chain, Respectively

Construction of Rap-hPAM4-Fd-DDD1-pdHL2

Rap-hPAM4-Fd-DDD1-pdHL2 is an expression vector for producing an a₂construct that comprises two identical Fab fusion proteins, eachcontaining ranpimase (Rap) and the DDD1 sequence linked to theN-terminus of the light chain and the C-terminus of the Fd chain,respectively. hPAM4 is a humanized monoclonal antibody specific forMUC-1. The plasmid vector Rap-hPAM4-γ1-pdHL2 used for producing theimmunotoxin referred to as 2L-Rap(N69Q)-hPAM4, which is composed of twomolecules of Rap, each fused to the N-terminus of the light chain ofhPAM4, was digested with Sac2 and NgoM4 to remove the fragment encodingthe CH1-CH3 domains, followed by ligation of the CH1-DDD1 fragment,which was excised from the plasmid vector C-DDD1-Fd-hMN-14-pdHL2 withSac2 and NgoM4 to generate Rap-hPAM4-Fd-DDD1-pdHL2.

Production, Purification and Characterization of Rap-hPAM4-Fab-DDD1

The Rap-hPAM4-Fd-DDD1-pdHL2 vector was transfected into NS0 myelomacells by electroporation. Rap-hPAM4-Fd-DDD1-pdHL2 is a di-cistronicexpression vector, which directs the synthesis and secretion of bothRap-fused hPAM4 light chain and hPAM4-Fd-DDD1, which combine to form theRap-Fab fusion protein. Each fusion protein forms a stable homodimer,referred to as Rap-hPAM4-Fab-DDD1, via the interaction of the DDD1domain.

Following electroporation, the cells were plated in 96-well tissueculture plates and transfectant clones were selected with 0.05 μMmethotrexate (MTX). Clones were screened for protein expression by ELISAusing microtitre plates coated with WS (a rat anti-id monoclonalantibody to hPAM4) and probed with ML98-1 (a mouse monoclonal antibodyto Rap) and HRP-conjugated goat anti-mouse Fc.

Rap-hPAM4-Fab-DDD1 was purified as described above using anAD1-C-affigel column. The initial productivity of the selected clone wasabout 0.5 mg per liter. SE-HPLC analysis (FIG. 17) of theaffinity-purified Rap-hPAM4-Fab-DDD1 shows a single protein peak with aretention time (8.15 min) consistent with the expected molecular mass of˜130 kDa. The binding affinity of Rap-hPAM4-Fab-DDD1 for WS was shown tobe similar to that of hPAM4 IgG (FIG. 18).

Example 4 Methods for Generating a₄ Constructs Composed of FourIdentical Fab Fusion Proteins, each Containing the DDD2 Sequence Linkedto the N-Terminus of the Fd Chain via a Peptide Spacer

Construction of N-DDD2-Fd-hMN-14-pdHL2

N-DDD2-Fd-hMN-14-pdHL2 is an expression vector for producing an a₄construct, referred to as the tetravalent N-DDD2-Fab-hMN-14 hereafter,that comprises four copies of a fusion protein in which the DDD2sequence is appended to hMN-14 Fab at the N-terminus of the Fd chain viaa flexible peptide spacer.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (DDD2 Top and DDD2 Bottom), whichcomprise residues 1-13 of DDD2, were made synthetically. Theoligonucleotides were annealed and phosphorylated with T4 polynucleotidekinase (PNK), resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases NcoI and PstI, respectively

DDD2 Top (SEQ ID NO: 15) 5′CATGTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3′ DDD2 Bottom (SEQ ID NO: 16)5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCA-3′

The duplex DNA was ligated with a vector fragment, DDD1-hMN14 Fd-SV3that was prepared by digestion with NcoI and PstI, to generate theintermediate construct DDD2-hMN14 Fd-SV3. A 1.28 kb insert fragment,which contained the coding sequence for DDD2-hMN14 Fd, was excised fromthe intermediate construct with XhoI and EagI restriction endonucleasesand ligated with hMN14-pdHL2 vector DNA that was prepared by digestionwith those same enzymes. The final expression vector isN-DDD2-Fd-hMN-14-pdHL2.

Production, Purification and Characterization of the TetravalentN-DDD2-Fab-hMN-14

N-DDD2-Fd-hMN-14-pdHL2 vector was transfected into Sp/EEE myeloma cellsby electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hMN-14 kappa light chain andN-DDD2-Fd-hMN-14, which combine to form the Fab-based subunitN-DDD2-Fab-hMN14. Following electroporation, the cells were plated in96-well tissue culture plates and transfectant clones were selected with0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WI2 (hMN-14 anti-Id) and detection was achieved withgoat anti-human Fab-HRP. The highest producing clones had an initialproductivity of approximately 10 mg/L. A total of 16 mg of N-DDD2-hMN-14was purified by protein L affinity chromatography from 1.8 liters ofroller bottle culture. Culture supernatants were concentratedapproximately 10-fold by ultrafiltration before loading onto a protein Lcolumn. The column was washed to baseline with PBS and N-DDD2-Fab-hMN14was eluted with 1 mM EDTA, 0.1 M NaAc, pH 2.5 and immediatelyneutralized with Tris-HCl. SE-HPLC analysis (FIG. 19) showed fourprotein peaks, two of which were subsequently attributed to thetetrameric a₄ (7.94 min) and dimeric a₂ (8.88 min) forms ofN-DDD2-Fab-hMN-14 and the remaining two were the dimer and monomer ofthe kappa chain. Most of the tetrameric a₄ form in the mixture wasconverted to the dimeric a₂ form (FIG. 20) upon adding a thiol reducingagent such as TCEP, suggesting that the tetrameric a₄ form apparently iscomposed of two dimeric a₂ structures linked through intermoleculardisulfide bridges formed between the cysteines present in DDD2. It isnoted that approximately 15% of the total N-DDD2-Fab-hMN-14 remains inthe a₄ form following reduction, even with high TCEP concentrations andlong reaction times, suggesting that other mechanisms such as domainswapping may contribute to the formation of the a₄ form, in addition todisulfide bridging. The tetravalent N-DDD2-Fab-hMN-14 was separated fromother molecular forms by gel filtration chromatography using aSuperdex-200 column.

Example 5 Methods for Generating a₄ Constructs Composed of FourIdentical Fab Fusion Proteins, each Containing the DDD2 Sequence Linkedto the C-Terminus of the Fd Chain via a Peptide Spacer

Construction of C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for producing an a₄construct, referred to as the tetravalent C-DDD2-Fab-hMN-14 hereafter,that comprises four copies of a fusion protein in which the DDD2sequence is appended to hMN-14 Fab at the C-terminus of the Fd chain viaa flexible peptide spacer.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG) and residues 1-13 of DDD2, weremade synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

G4S-DDD2 top (SEQ ID NO: 17)5′GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3′ G4S-DDD2 bottom (SEQ ID NO: 18)5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTCCGC CAGACCCGCCACCTCCG-3′

The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, whichwas prepared by digestion with BamHI and PstI, to generate the shuttlevector CH1-DDD2-pGemT. A 507 bp fragment was excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vectorhMN14(I)-pdHL2, which was prepared by digestion with SacII and EagI. Thefinal expression construct is C-DDD2-Fd-hMN-14-pdHL2.

Construction of C-DDD2-Fd-hA20-pdHL2

C-DDD2-Fd-hA20-pdHL2 is an expression vector for producing an a₄construct, referred to as the tetravalent C-DDD2-Fab-hA20 hereafter,that comprises four copies of a fusion protein in which the DDD2sequence is appended to hA20-Fab at the C-terminus of the Fd chain via aflexible peptide spacer. hA20 is a humanized monoclonal antibodyspecific for CD20.

The expression vector was engineered in three steps as follows. First,the expression vector hA20-IgG-pdHL2 was digested with Sac2 and NdeI toyield the 7578-bp fragment. Next, the expression vectorC-DDD2-hMN-14-Fd-pdHL2 was digested with Sac2 and NdelI and the 509-bpfragment coding for CH1-DDD2 was isolated. Third, the 7578-bp fragmentwas ligated with the 509-bp fragment to generate C-DDD2-Fd-hA20-phHL2.

Construction of C-DDD2-Fd-hMN-3-pdHL2

C-DDD2-Fd-hMN3-pdHL2 is an expression vector for producing an a₄construct, referred to as the tetravalent C-DDD2-Fab-hMN-3 hereafter,that comprises four copies of a fusion protein in which the DDD2sequence is appended to hMN3-Fab at the C-terminus of the Fd chain via aflexible peptide spacer. hMN-3 is a humanized monoclonal antibodyspecific for the N domain of CEA (CEACAM5) or NCA-90 (CEACAM6).

The expression vector was engineered in three steps as follows. First,the expression vector hMN-3-IgG-pdHL2 was digested with Sac2 and NgoM4to yield the 8118-bp fragment. Next, the expression vectorC-DDD2-hMN-14-Fd-pdHL2 was digested with Sac2 and NgoM4 and the 509-bpfragment coding for CH1-DDD2 was isolated. Third, the 8118-bp fragmentwas ligated with the 509-bp fragment to generate C-DDD2-Fd-hMN-3-phHL2.

Construction of C-DDD2-Fd-hLL2-pdHL2

C-DDD2-Fd-hLL2-pdHL2 is an expression vector for producing an a₄construct, referred to as the tetravalent C-DDD2-Fab-hLL2 hereafter,that comprises four copies of a fusion protein in which the DDD2sequence is appended to hLL2-Fab at the C-terminus of the Fd chain via aflexible peptide spacer. hLL2 is a humanized monoclonal antibodyspecific for CD22.

The expression vector was engineered in three steps as follows. First,the expression vector hLL2-IgG-pdHL2 was digested with Sac2 and NdeI toyield the 7578-bp fragment. Next, the expression vectorC-DDD2-hMN-14-Fd-pdHL2 was digested with Sac2 and NdeI and the 509-bpfragment coding for CH1-DDD2 was isolated. Third, the 7578-bp fragmentwas ligated with the 509-bp fragment to generate C-DDD2-Fd-hLL2-phHL2.

Production, Purification and Characterization of the TetravalentC-DDD2-Fab-hMN-14

C-DDD2-Fd-hMN-14-pdHL2 vector was transfected into Sp/EEE myeloma cellsby electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hMN-14 kappa light chain andC-DDD2-Fd-hMN-14, which combine to form C-DDD2-Fab-hMN14. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WI2 (hMN-14 anti-Id) and detection was achieved withgoat anti-human Fab-HRP. The highest producing clones had an initialproductivity of approximately 10 mg/L, which was 10-fold higher thanthat of N-DDD2-Fab-hMN-14. A total of 200 mg of C-DDD2-Fab-hMN-14 waspurified by protein L affinity chromatography from 1.8 liters of rollerbottle culture as described above for N-DDD2-Fab-hMN-14. The SE-HPLCprofile of the Protein L-purified C-DDD2-Fab-hMN-14 was similar to thatof N-DDD2-Fab-hMN-14, showing four protein peaks. Two of the fourprotein peaks were attributed to the tetrameric a₄ (8.40 min) anddimeric a₂ (9.26 min) forms of C-DDD2-Fab-hMN-14 and the remaining tworepresent dimer and monomer of the kappa chain. The tetravalentC-DDD2-Fab-hMN-14 was separated from other molecular forms by gelfiltration chromatography using a SUPERDEX®-200 column. LikeN-DDD2-Fab-hMN-14, addition of TCEP converts most of the a₄ form to thea₂ form, as illustrated in FIG. 21. The SE-HPLC profile of thetetravalent C-DDD2-Fab-hMN-14 on a tandem column system is shown in FIG.22, appearing as a single peak with a retention time of 19.57min. Theability of the tetravalent C-DDD2-Fab-hMN-14 to bind to four WI2fragments is shown in FIG. 23.

Production, Purification and Characterization of the TetravalentC-DDD2-Fab-hA20

C-DDD2-Fd-hA20-pdHL2 vector was transfected into NS0 myeloma cells byelectroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hA20 kappa light chain andC-DDD2-Fd-hA20, which combine to form C-DDD2-Fab-hA20. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WR2 (a rat anti-id to hA20) and detection wasachieved with goat anti-human Fab-HRP. The highest producing clones hadan initial productivity of approximately 10 mg/L. The tetravalentC-DDD2-Fab-hA20 was purified from cell culture supernatants produced inroller bottles by Protein L affinity chromatography followed bySUPERDEX®-200 gel filtration. The SE-HPLC profile of the tetravalentC-DDD2-Fab-hA20 is shown in FIG. 24. The tetravalent C-DDD2-Fab-hA20showed potent anti-proliferative activity on Daudi and Ramos even in theabsence of anti-IgM (FIG. 25). By contrast, the bivalent hA20 IgG orF(ab′)2 was inactive in inhibiting the growth of Daudi or Ramos underthe same conditions either in the absence or presence of anti-IgM. Theobserved anti-proliferative activity of hA20 IgG or F(ab′)2 in thepresence of anti-IgM was apparently due to that of anti-IgM.

Production and Purification of the Tetravalent C-DDD2-Fab-hMN-3

C-DDD2-Fd-hMN-3-pdHL2 vector was transfected into NS0 myeloma cells byelectroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hMN-3 kappa light chain andC-DDD2-Fd-hMN-3, which combine to form C-DDD2-Fab-hMN-3. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with CEACAM5 and detection was achieved with goatanti-human Fab-HRP. The highest producing clones had an initialproductivity of approximately 10 mg/L. The tetravalent C-DDD2-Fab-hMN-3was purified from cell culture supernatants produced in roller bottlesby Protein L affinity chromatography followed by Superdex-200 gelfiltration.

Production and Purification of the Tetravalent C-DDD2-Fab-hLL2

C-DDD2-Fd-hLL2-pdHL2 vector was transfected into Sp2/0-derived myelomacells by electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hLL2 kappa light chain andC-DDD2-Fd-hLL2, which combine to form C-DDD2-Fab-hLL2. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WN (a rat anti-id to hLL2) and detection was achievedwith goat anti-human Fab-HRP. The highest producing clones had aninitial productivity of approximately 15 mg/L. The tetravalentC-DDD2-Fab-hLL2 was purified from cell culture supernatants produced inroller bottles by Protein L affinity chromatography followed bySUPERDEX®-200 gel filtration.

Example 6 Methods for Generating a₂a′₂ Constructs from Two Distinct a₄and a′₄ Constructs

Production, Purification and Characterization of the BispecificTetravalent C-DDD2-Fab-hMN-3 x C-DDD2-Fab-hA20.

The tetravalent C-DDD2-Fab-hMN-3 and the tetravalent C-DDD2-Fab-hA20obtained from Example 5 were combined and reduced with 1 mM glutathioneat RT for 1 h followed by adding oxidized glutathione to a finalconcentration of 2 mM. The tetrameric fraction was purified from theother molecular forms by gel filtration on a SUPERDEX®-200 column. Theformation of the bispecific tetravalent C-DDD2-Fab-hMN-3 xC-DDD2-Fab-hA20 was demonstrated by ELISA using plates coated withCEACAM5 and probed with WR2, as shown in FIG. 26.

Production, Purification and Characterization of the BispecificTetravalent C-DDD2-Fab-hMN-3 x C-DDD2-Fab-hMN-14

The tetravalent C-DDD2-Fab-hMN-3 and the tetravalent C-DDD2-Fab-hMN-14obtained from Example 5 were combined and reduced with 1 mM glutathioneat RT for 1 h followed by adding oxidized glutathione to a finalconcentration of 2 mM. The tetrameric fraction was purified from theother molecular forms by gel filtration on a SUPERDEX®-200 column. Theformation of the bispecific tetravalent C-DDD2-Fab-hMN-3 xC-DDD2-Fab-hMN-14 was demonstrated by flow cytometry using BXPC3 cellsas shown in FIG. 27.

Production and Purification of the Bispecific TetravalentC-DDD2-Fab-hA20 x C-DDD2-Fab-hLL2

The tetravalent C-DDD2-Fab-hA20 and the tetravalent C-DDD2-Fab-hLL2obtained from Example 5 were combined and reduced with 1 mM glutathioneat RT for 1 h followed by adding oxidized glutathione to a finalconcentration of 2 mM. The tetrameric fraction was purified from theother molecular forms by gel filtration on a Superdex-200 column. Theformation of the bispecific tetravalent C-DDD2-Fab-hA20 xC-DDD2-Fab-hLL2 was demonstrated by ELISA using plates coated with WN (arat anti-id to hLL2) and probed withWR2 (a rat anti-id to hA20).

TABLE 1 Selected Examples of Type I Products for which the subunits of a₂ are based on binding domains derived from immunoglobulins TargetApplication X Treating or detecting a disease bearing the X marker CD14Treating septic shock CD111/nectin-1 Treating herpesvirus infectionFolate receptor α Treating filovirus infection (e.g. Ebola and Marburgviruses) gp120 Treating HIV-1/AIDS IL-6 Treating myeloma, arthritis andother autoimmune disease IL-5 Treating asthma IL-8 Treating generalinfection CD154 Treating lupus, transplant rejection, AID IgE Treatingasthma as indicated by Xolair ® LFA-1 Treating transplant rejection CD3Treating transplant rejection as indicated by OKT3 ® β-tryptase Treatingallergy, inflammation CD105/endoglin Anti-angiogenesis GpIIb/IIaTreating thrombosis as indicated by RepPro ™ TNF-α Treating arthritis asindicated by HUMIRA ™ or REMICADE ® RSV F-protein RSV therapy asindicated by Synagis ™ A1B1 of CEA Inhibitingadhesion/invasion/metastasis of solid cancers N domain of CEA Inhibitingadhesion/invasion/metastasis of solid cancers Pgp/p-170 Reversingmultiple drug resistance VEGF Neutralizing VEGF Placenta growthNeutralizing factor (PlGF) VEGFR1/Flt-1 Treating cancers Blys/CD257Treating lupus and arthritis APRIL/CD256 Treating lupus and arthritis

TABLE 2 Selected Examples of Type 2 Products for which the subunits of a₂ are based on nonimmunoglobulin proteins Precursor Application SolubleTumor necrosis Treating arthritis as indicated by Enbrel ® factorreceptor (sTNFR) sTNFR-VL-CL Treating arthritis as indicated by Enbrel ®sTNFR-CH2-CH3 Treating arthritis as indicated by Enbrel ® Ranpirnase(Rap) Treating cancers Rap-VL-CL Treating cancers Rap-CH2-CH3 Treatingcancers Tissue plasminogen Treating diseases as indicated by Activase ®activator (tPA) tPA-VL-CL Treating diseases as indicated by Activase ®tPA-CH2-CH3 Treating diseases as indicated by Activase ® Erythropoietin(EPO) Treating anemia as indicated by Epogen ® EPO-VL-CL Treating anemiaas indicated by Epogen ® EPO-CH2-CH3 Treating anemia as indicated byEpogen ® Thrombopoietin (TPO) Treating thrombocytopenia TPO-VL-CLTreating thrombocytopenia TPO-CH2-CH3 Treating thrombocytopeniaInterlukin (IL)-11 Treating thrombocytopenia as indicated by Neumega ®IL-11-VL-CL Treating thrombocytopenia as indicated by Neumega ®IL-11-CH2-CH3 Treating thrombocytopenia as indicated by Neumega ®Granulocyte-colony Treating neutropenia as indicated by stimulatingfactor (G-CSF) Neupogen ® G-CSF-VL-CL Treating neutropenia as indicatedby Neupogen ® G-CSF-CH2-CH3 Treating neutropenia as indicated byNeupogen ® Interferon (IFN)-α2 Treating hepatitis as indicated by IntronA ® IFN-α2-VL-CL Treating hepatitis as indicated by Intron A ®IFN-α2-CH2-CH3 Treating hepatitis as indicated by Intron A ® IFN-β1Treating multiple sclerosis as indicated by Betaseron ® IFN-β1-VL-CLTreating multiple sclerosis as indicated by Betaseron ® IFN-β1-CH2-CH3Treating multiple sclerosis as indicated by Betaseron ® Coagulationfactor IX Treating hemophilia B as indicated by BeneFix ™ Coagulationfactor Treating hemophilia B as indicated by IX-VL-CL BeneFix ™Coagulation Treating hemophilia B as indicated by factor-IX-CH2-CH3BeneFix ™ GM-CSF Treating diseases as indicated by Leukine ®GM-CSF-VL-CL Treating diseases as indicated by Leukine ® GM-CSF-CH2-CH3Treating diseases as indicated by Leukine ® PlGF antagonist peptidesNeutralizing VEGF antagonist peptides Neutralizing VEGF Tyrosine kinaseinhibitors Treating cancers Aβ12-28P fused to Treating Alzheimer′sdisease CH2-CH3

TABLE 3 Selected Examples of Type 3 Products for which the subunits of a₄ are based on binding domains derived from immunoglobulins TargetApplication X Treating or detecting a disease bearing the X marker CD14Treating septic shock CD111/nectin-1 Treating herpes simplex virusinfection Folate receptor α Treating Filovirus infection (e.g. Ebola andMarburg viruses) gp120 Treating HIV-1/AIDS IL-6 Treating myeloma,arthritis and other autoimmune disease IL-5 Treating asthma IL-8Treating general infection CD154 Treating lupus, transplant rejection,AID IgE Treating asthma as indicated by Xolair ® LFA-1 Treatingtransplant rejection β-tryptase Treating allergy, inflammationCD105/endoglin Anti-angiogenesis GpIIb/IIa Treating thrombosis asindicated by RepPro ™ TNF-α Treating arthritis as indicated by HUMIRA ™or REMICADE ® RSV F-protein RSV therapy as indicated by Synagis ™ A1B1of CEA Inhibiting adhesion/invasion/metastasis of solid cancers N domainof Inhibiting adhesion/invasion/metastasis of solid cancers CEA CD20Treating B-cell lymphomas or autoimmune diseases, as indicated byRituxan ™ CD22 Treating B-cell lymphomas or autoimmune diseases CD19Treating B-cell lymphoma or autoimmune diseases CD80 Lymphoma therapyHLA-DR Treating cancers or autoimmune diseases CD74 Treating cancers orautoimmune diseases MUC1 Treating cancers HER2/neu Treating cancers EGFRTreating cancers Insulin-like Treating cancers growth factor MIFTreating autoimmune diseases CD83 Treating autoimmune diseases CD3Treating transplant rejection as indicated by OKT3 ® IL-2Rα/CD25Preventing kidney transplant rejection as indicated by Zenapax ® orSimulect ® ICAM-1 Preventing human rhinovirus infection Pgp/p-170Reversing multiple drug resistance VEGF Neutralizing VEGF PlGFNeutralizing VEGFR1/Flt-1 Treating cancers Blys/CD257 Treating lupus andarthritis April/CD256 Treating lupus and arthritis

TABLE 4 Selected Examples of Type 4 Products for which the subunits of a₄ are based on nonimmunoglobulin proteins Precursor Application sTNFRTreating arthritis as indicated by Enbrel ® sTNFR-VL-CL Treatingarthritis as indicated by Enbrel ® sTNFR-CH2-CH3 Treating arthritis asindicated by Enbrel ® Rap Treating cancers Rap-VL-CL Treating cancersRap-CH2-CH3 Treating cancers tPA Treating diseases as indicated byActivase ® tPA-VL-CL Treating diseases as indicated by Activase ®tPA-CH2-CH3 Treating diseases as indicated by Activase ® EPO Treatinganemia as indicated by Epogen ® or Aranesp ® EPO-VL-CL Treating anemiaas indicated by Epogen ® or Aranesp ® EPO-CH2-CH3 Treating anemia asindicated by Epogen ® or Aranesp ® TPO Treating thrombocytopeniaTPO-VL-CL Treating thrombocytopenia TPO-CH2-CH3 Treatingthrombocytopenia IL-11 Treating thrombocytopenia as indicated byNeumega ® IL-11-VL-CL Treating thrombocytopenia as indicated byNeumega ® IL-11 -CH2-CH3 Treating thrombocytopenia as indicated byNeumega ® G-CSF Treating neutropenia as indicated by Neupogen ®G-CSF-VL-CL Treating neutropenia as indicated by Neupogen ®G-CSF-CH2-C1-13 Treating neutropenia as indicated by Neupogen ® IFN-α2Treating hepatitis as indicated by Intron A ® IFN-α2-VL-CL Treatinghepatitis as indicated by Intron A ® IFN-α2-CH2-C113 Treating hepatitisas indicated by Intron A ® IFN-β1 Treating multiple sclerosis asindicated by Betaseron ® IFN-β1-VL-CL Treating multiple sclerosis asindicated by Betaseron ® IFN-β1 -CH2-CH3 Treating multiple sclerosis asindicated by Betaseron ® Coagulation factor IX Treating hemophilia B asindicated by BeneFix ™ Coagulation Treating hemophilia B as indicated byfactor IX-VL-CL BeneFix ™ Coagulation Treating hemophilia B as indicatedby factor-IX-CH2-CH3 BeneFix ™ GM-CSF Treating diseases as indicated byLeukine ® GM-CSF-VL-CL Treating diseases as indicated by Leukine ®GM-CSF-CH2-CH3 Treating diseases as indicated by Leukine ® PlGFantagonist peptides Neutralizing PlGF or Flt-1, also treating cancersVEGF antagonist peptides Neutralizing VEGF, also treating cancersTyrosine kinase inhibitors Treating cancers Aβ12-28P fused TreatingAlzheimer′s disease to CH2-CH3

TABLE 5 Selected Examples of Type 5 products for which the subunits of a₂ a′ ₂ are based on binding domains of two different immunoglobulinsTarget 1 Target 2 Application CD20 CD22 Treating lymphomas or autoimmunediseases CD19 CD20 Treating lymphomas or autoimmune diseases EGFR IGFR1Treating solid tumors VEGFR1/Flt-1 VEGFR2/KDR Blocking VEGF bindingVEGFR3/Flt-4 VEGFR2/KDR Blocking VEGF binding CD19 CD3/TCR Treatingcancers CD19 CD16/FcγRIIIa Treating cancers CD19 CD64/FcγRI Treatingcancers HER2/neu CD89/FcαRI Treating cancers HER2/neu CD16 Treatingcancers HER2/neu CD64 Treating cancers HER2/neu CD3 Treating cancersHER2 (Herceptin) HER2 (Omnitarg) Treating cancers HER2 HER3 Treatingcancers CD30 CD64 Treating cancers CD33 CD64 Treating cancers EGFR CD2Treating cancers EGFR CD64 Treating cancers EGFR CD16 Treating cancersEGFR CD89 Treating cancers PfMSP-1 CD3 Treating malaria EpCAM/17-1A CD3Treating cancers hTR CD3 Treating cancers IL-2R/Tac CD3 Treating cancersCA19-9 CD16 Treating cancers MUC1 CD64 Treating cancers HLA class IICD64 Treating cancers G _(D2) CD64 Treating neuroblastoma Carbonicanhydrase IX CD89 Treating renal cell carcinoma TAG-72 CD89 Treatingcancers EpCAM Adenovirus fiber knob Retargeting viral vector to EpCAM +cancers PSMA Adenovirus fiber knob Retargeting viral vector to prostatecancers CEA Adenovirus fiber knob Retargeting viral vector toCEA-positive cancer HMWMAA Adenovirus fiber knob Retargeting viralvector to melanoma Carbonic anhydrase IX Adenovirus fiber knobRetargeting viral vector to renal cell carcinoma CD40 Adenovirus fiberknob Retargeting viral vector to dendritic cells M13 coat proteinAlkaline phosphatase Detecting virus GpIIb/IIIa tPA Enhancingthrombolysis A1B1 of CEA N of CEA Inhibiting cancer invasion/metastasisCD20 CD55 Treating B-cell lymphoma CD20 CD59 Treating B-cell lymphomaCD20 CD46 Treating B-cell lymphoma Carbonic anhydrase IX CD55 Treatingrenal cell carcinoma EpCAM CD55 Treating cancers Migration inhibitoryLipopolysaccharide Treating sepsis and septic factor (MIF) (LPS) shockMIF C5a receptor (C5aR) Treating sepsis and septic shock MIF IL-6Treating sepsis and septic shock Toll-like receptor-2 LPS Treatingsepsis and septic (TLR2) shock High mobility group TNF-α Treating sepsisand septic box protein 1 shock (HMGB-1) MIF NCA-90/CEACAM6 Treatingcancer, sepsis and septic shock MIF HLA-DR Treating sepsis and septicshock MIF Low-density Treating atherosclerosis lipoprotein (LDL) NCA90LDL Treating atherosclerosis CD83 LDL Treating atherosclerosis CD74 LDLTreating atherosclerosis TNF CD20 Lymphoma therapy TNF CD22 Lymphomatherapy TNF CD74 Treating cancers TNF MIF Treating autoimmune diseasesTNF CD83 Treating autoimmune diseases Tumor antigens Histamine-succinly-Pre-targeting applications glycine (HSG) for cancer diagnosis andtherapy Blys/CD257 April/CD256 Treating lupus and arthritis

TABLE 6 Selected Examples of Type 6 products for which the subunits of a₂ a′ ₂ are based on immunoglobulins and non-immunoglobulins Target forPrecursor for mAb nonimmunoglobulin Application CD74 Rap-VL-CL Treatingcancers CD22 Rap-VL-CL Treating cancers MUC1 Rap-VL-CL Treating cancersEGP-1 Rap-VL-CL Treating cancers IGF1R Rap-VL-CL Treating cancersPgp/p-170 Rap-VL-CL Treating cancers CD22 Pseudomonas exotoxin Treatingcancers (PE)38 CD30 PE38 Treating cancers CD25/Tac PE38 Treating cancersLe ^(Y) PE38 Treating cancers Mesothelin PE38 Treating cancers HER2 PE38Treating cancers EpCAM PE38 Treating cancers Pgp/p-170 PE38 Treatingcancers CD25 dgA Treating cancers CD30 dgA Treating cancers CD19 dgATreating cancers CD22 dgA Treating cancers CD25 PLC Treating cancersGp240 Gelonin Treating melanoma Pgp/p-170 IL-2 Treating cancers CD3DT390 Treating graft versus host disease (GVHD) GpIIb/IIIa tPA Enhancingthrombolysis GpIIb/IIIa urokinase Enhancing thrombolysis GpIIb/IIIahirudin Enhancing thrombolysis X Carboxypeptidase G2 (CPG2) Prodrugtherapy X penicillinamidase Prodrug therapy X β-lactamase Prodrugtherapy X Cytosine deaminase Prodrug therapy X Nitroreductase Prodrugtherapy Aβ Tf Treating Alzheimer′s disease

TABLE 7 Selected Examples of Type 7 products for which the subunits of a₂ a′ ₂ are based on two different non-immunoglobulins Precursor 1Precursor 2 Application IL-4 PE38 Treating pancreatic cancer IL-4Rap-VL-CL Treating pancreatic cancer sIL-4R sIL-13R Treating asthma,allergy Aβ12-28P fused to CH2-CH3 Tf Treating Alzheimer′s diseaseAβ12-28P Tf Treating Alzheimer′s disease

1. A method of delivering a diagnostic or therapeutic agent comprising:a) obtaining a homodimer, each monomer of the homodimer comprising adimerization and docking domain (DDD) of human protein kinase Aregulatory subunit wherein the DDD consists the sequence of SEQ ID NO: 1(DDD1) or SEQ ID NO: 2 (DDD2) and, wherein the DDD peptide sequencesbind together to form a homodimer and the homodimer comprises at leastone diagnostic or therapeutic agent; and b) administering the homodimerto a subject.
 2. The method of claim 1, wherein the two DDD peptidesequences of the homodimer are covalently attached to each other bydisulfide bonds.
 3. The method of claim 1, wherein each monomer furthercomprises an antigen-binding antibody fragment that binds to atumor-associated antigen (TAA).
 4. The method of claim 3, wherein eachmonomer is a fusion protein comprising said DDD peptide sequence of SEQID NO: 2 and an antigen-binding antibody fragment.
 5. The method ofclaim 4, wherein the monomer further comprises a linker peptide betweenthe antigen-binding antibody fragment and the DDD.
 6. The method ofclaim 3, wherein the antigen-binding antibody fragment is chemicallylinked to the DDD peptide sequence.
 7. The method of claim 3, whereinthe antigen-binding antibody fragment is selected from the groupconsisting of a Fab fragment, a Fab′ fragment, a single domain antibody(DAB) and a single chain Fv (scFv).
 8. The method of claim 3, whereinthe antigen-binding antibody fragment binds to a tumor-associatedantigen (TAA) and the therapeutic agent is an anti-cancer therapeuticagent.
 9. The method of claim 8, wherein the tumor-associated antigen isselected from the group consisting of carbonic anhydrase IX,alpha-fetoprotein, antigen specific for A33 antibody, BrE3-antigen,CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23,CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specificantigen-p (CSAp), CEA (CEACAM5), CEACAM6, EGFR, EGP-1, EGP-2, Ep-CAM,Flt-1, Flt-3, folate receptor, G250 antigen, HLA-DR, human chorionicGonadotropin (HCG) and its subunits, HER2/neu, hypoxia inducible factor(HIF-1), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage migration inhibitoryfactor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigenspecific for PAM-4 antibody, placental growth factor, p53, prostaticacid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAILreceptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGF, ED-B fibronectin, and 17-1A-antigen.
 10. The method ofclaim 8, wherein the antigen-binding antibody fragment is a fragment ofan antibody selected from the group consisting of anti-CEA (hMN-14),anti-CD20 (hA20), anti-CD22 (hLL2), anti-CD74 (hLL1), anti-MUC-1(hPAM4), anti-CD14, anti-CD111, omalizumab, muromonab, abciximab,infliximab and palivizumab.
 11. The method of claim 3, wherein theantigen-binding antibody fragment comprises an immunoglobulin lightchain (VL-CL) or an immunoglobulin Fc domain (CH2-CH3).
 12. The methodof claim 11, wherein the cysteine at the carboxyl-terminus of the CLthat connects the CL to CH1 is deleted or mutated to a non-cysteine. 13.The method of claim 3, wherein the antigen-binding antibody fragment isa fragment of a human, humanized or chimeric antibody.
 14. The method ofclaim 1, wherein the therapeutic agent is selected from the goupconsisting of a chemotherapeutic agent, a cytokine, a chemokine, ananti-angiogenic agent, an apoptotic agent, a drug, a prodrug, a toxin,an enzyme, a radioisotope, an immunomodulator, an antibiotic and ahormone.
 15. The method of claim 14, wherein the drug is selected fromthe group consisting of abrin, amantadine, amoxicillin, amphotericin B,ampicillin, aplidin, azaribine, anastrozole, azacytidine, aztreonam,azithromycin, bacitracin, trimethoprim/sulfamethoxazole, Batrafen,bifonazole, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carbenicillin,caspofungin, carmustine, cefaclor, cefazolin, cephalosporins, cefepime,ceftriaxone, cefotaxime, celecoxib, chlorambucil, chloramphenicol,ciprofloxacin, cisplatin, irinotecan (CPT-11), SN-38, carboplatin,cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel,dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone,diethylstilbestrol, diphtheria toxin, DNase I, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), doxycycline, cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, estrogen receptor binding agents, etoposide,etoposide glucuronide, etoposide phosphate, erythrocycline,erythromycin, flagyl, farnesyl-protein transferase inhibitors,floxuridine (FUdR), 3′,5′—O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, fluorouracil, fluoxymesterone, ganciclovir, gentamycin,gelonin, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, isoniazid, itraconazole, kanamycin,ketoconazole, L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, minocycline, naftifine, nalidixic acid, neomycin, navelbine,nitrosurea, nystatin, ranpirnase, oxacillin, paromomycin, penicillin,pentamidine, piperacillin-tazobactam, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, pokeweed antiviral protein,PSI-341, semustine, rifabutin, rifampin, rimantadine, streptomycin,sulfamethoxazole, sulfasalazine, streptozocin, tamoxifen, taxanes,taxol, testosterone propionate, tetracycline, thalidomide, thioguanine,thiotepa, teniposide, topotecan, transplatinum, trimethoprimsulfamethoxazole, uracil mustard, valacyclovir, vancomycin, vinblastine,vinorelbine, vincristine, zanamir and zithromycin.
 16. The method ofclaim 15, wherein the toxin is selected from the group consisting of abacterial toxin, a plant toxin, ricin, abrin, a ribonuclease (RNase),DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin,Ranpirnase (Rap) and Rap (N69Q).
 17. The method of claim 14, wherein theimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin an interleukin, acolony-stimulating factor, interferon-α, interferon-β, interferon-γ, andthe stem cell growth factor designated “S1 factor.”
 18. The method ofclaim 14, wherein the cytokine is selected from the group consisting ofhuman growth hormone, N-methionyl human growth hormone, bovine growthhormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin,prorelaxin, follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH), hepatic growth factor,prostaglandin, fibroblast growth factor, prolactin, placental lactogen,OB protein, tumor necrosis factor-α, tumor necrosis factor-β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,erythropoietin (EPO), osteoinductive factor, interferon-α, interferon-β,interferon-γ, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF(GM-CSF), granulocyte-CSF (G-CSF), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, LIF, kit-ligand, FLT-3, angiostatin,thrombospondin, endostatin and LT.
 19. The method of claim 1, whereinthe diagnostic or therapeutic agent is selected from the groupconsisting of ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁴⁵Ti, ⁵⁷Co,⁵⁸Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁶⁶Dy, ¹⁵²Eu, ¹⁸F, ⁶⁷Ga, ⁶⁸Ga, ^(195m)Hg, ¹⁶⁶Ho,³H, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁵²Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁹¹Os, ²¹²Pb, ³²P,³³P, ¹⁴²Pr, ^(195m)Pt, ²²³Ra, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ⁴⁷Sc, ⁷⁵Se, ¹¹¹Ag,¹⁵³Sm, ⁸⁹Sr, ³⁵S, ¹⁶¹Tb, ^(94m)Tc, ^(99m)Tc, ⁸⁶Y, ⁹⁰Y, and ⁸⁹Zr.
 20. Themethod of claim 1, wherein the diagnostic agent is selected from thegroup consisting of a radioisotope, an imaging agent, a dye, an enzyme,a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, aparamagnetic ion and an ultrasound label.
 21. The method of claim 20,wherein the imaging agent is selected from the group consisting ofchromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) erbium (III), lanthanum (III), gold (III), lead (II) andbismuth (III).
 22. The method of claim 20, wherein the fluorescent agentis selected from the group consisting of Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole),Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, phthalocyanines, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red.
 23. The method of claim 1, whereinthe homodimer further comprises one or more effectors or carriersconjugated to the homodimer by either covalent or non-covalent linkage.24. The method of claim 23, wherein the effector is selected from thegroup consisting of a diagnostic agent, a therapeutic agent, achemotherapeutic agent, a radioisotope, an imaging agent, ananti-angiogenic agent, a cytokine, a chemokine, a growth factor, a drug,a prodrug, an enzyme, a ligand for a cell surface receptor, a chelator,an immunomodulator, a hormone, a photodetectable label, a dye, a toxin,a contrast agent, a paramagnetic label, an ultrasound label, apro-apoptotic agent, a liposome and a nanoparticle.
 25. The method ofclaim 23, wherein the effectors or carriers are chemically cross-linkedto the homodimer.
 26. The method of claim 23, wherein the homodimer isattached to two or more effectors or two or more carriers.
 27. Themethod of claim 26, wherein the two or more carriers or two or moreeffectors are either identical or different.
 28. The method of claim 23,wherein the one or more carriers comprise at least one diagnostic ortherapeutic agent.